Stable plurilamellar vesicles

ABSTRACT

A new and substantially improved type of lipid vesicle, called stable plurilamellar vesicles (SPLVs), are described, as well as the process for making the same and X-ray diffraction methods for identifying the same. SPLVs are characterized by lipid bilayers enclosing aqueous compartments containing one or more entrapped solutes, the concentration of such solutes in each aqueous compartment being substantially equal to the emunization of solutes used to prepare the SPLVs. The bilayers of SPLVs are substantially non-compressed. SPLVs are stable during storage and can be used in vivo for the sustained release of compounds and in the treatment of disease.

This is a division of application Ser. No. 660,573, filed Oct. 12, 1984,now U.S. Pat. No. 5,030,453, which is a continuation-in-part of acopending application Ser. No. 633,481, filed Jul. 26, 1984 now U.S.Pat. No. 5,000,958 and a continuation-in-part of copending applicationSer. No. 476,496, filed Mar. 24, 1983 now U.S. Pat. No. 4,522,803 and acontinuation-in-part of copending application Ser. No. 521,176, filedAug. 8, 1983 now U.S. Pat. No. 4,588,578.

TABLE OF CONTENTS

1. Field of the Invention

2. Background of the Invention

2.1. Liposomes

2.2. Uses of Liposomes

3. Summary of the Invention

4. Brief Description of the Figures

5. Detailed Description of the Invention

5.1. Preparation of SPLVs

5.1.1. Monophasic Solvent System Process

5.1.2. Emulsification Process

5.1.3. SPLV Constituents

5.2. Characterization of SPLVs

5.2.1. Stability of SPLVs in Storage

5.2.2. Stability of SPLVs in Other Environments

5.2.3. Entrapment of Active Material by SPLVs

5.2.4. Effect of Varying the Initial Lipid to Aqueous Ratio

5.2.5. Volume of SPLVs

5.2.6. Buoyant Density of SPLVs

5.2.7. Osmotic Properties of SPLVs

5.2.8. Electron Spin Resonance

5.2.9. X-ray Diffraction

5.2.9.1. X-ray Diffraction Methods

5.2.9.2. X-ray Diffraction Signatures

5.2.9.3. Long Spacing Signature

5.2.9.4. Relation of Long Spacing Signature to the Osmotic Properties ofSPLVs and MLVs

5.2.9.5. Solute Distribution in SPLVs vs. MLVs

5.2.9.6. Identification of SPLVs

5.2.9.7. Interpretation of Results

5.3. Uses of SPLVs

5.3.1. Delivery of Bioactive Compounds

5.3.1.1. Delivery In Vitro

5.3.1.2. Delivery in Vivo

5.3.2. Treatment of Pathologies

6. Example: Preparation of SPLVs by the Monophasic Solvent SystemProcesses

6.1. SPLVs Containing Tetracyclines

6.2. SPLVs Containing Gentamicin and Nafcillin

6.3. SPLVs Containing Gentamicin

6.4. SPLVs Containing Chloramphenicol

6.5. SPLVs Containing Tobramycin

6.6. SPLVs Containing Ticarcillin

6.7. SPLVs Containing Ticarcillin and Tobramycin

6.8. Alternative Methods of Preparing SPLVs

6.9. Preparation of SPLVs Using Various Solvent System

7. Preparation of SPLVs by the Emulsification Processes

7.1. SPLVs Containing Antibiotics

7.2. Preparation of SPLVs Containing Gentamicin or Nafcillin

7.3. Preparation of SPLVs Containing Both Gentamicin and Nafcillin

7.4. SPLVs Containing Gentamicin and Clindamycin

7.5. SPLVs Containing Other Membrane Constituents

7.6. SPLVs Containing Pilocarpine

7.7. SPLVs Prepared With and Without BHT

8. Example: SPLV Mediated Delivery In Vitro

9. Example: Treatment of Intracellular Infections

9.1. Effect of a Single Treatment of B. Canis Infection UsingSPLV-entrapped Antibiotic

9.2. Effect of Multiple Treatment of B. Canis Infection UsingSPLV-entrapped Antibiotic

9.3. Effectiveness of Treatments Using MLVs as Compared to SPLVs

9.4. Effect of Various SPLV-entrapped Antibiotics On Treatment ofInfection

9.5. Treatment of Dogs Infected with B. Canis

9.6. Treatment of B. Abortus in Guinea Pigs

9.7. Treatment of B. Abortus Infection in Cows

10. Example: Treatment of Systemic Infection

10.1. Effect of Single Treatment of S. Typhimurium Infection UsingSPLV-entrapped Antibiotics

10.2. Effect of Multiple Treatment of S. Typhimurium Infection UsingSPLV-entrapped Antibiotics

10.3. Example: Enhancement of Antibacterial Activity in TreatingSalmonella Typhimurium Infections Using SPLVs Containing Gentamicin andNafcillin

10.4. Example: Enhancement of Antibacterial Activity in TreatingSalmonellosis Using SPLVs Containing Gentamicin and Nafcillin

10.5. Example: Enhancement of Antibacterial Activity in TreatingSalmonella Typhimurium Infections Using SPLVs Containing Gentamicin andNafcillin

11. Example: Treatment of Ocular Afflictions

11.1. Treatment of Infectious Keratoconjunctivitis in Mice

11.2. Treatment of Rabbit Conjunctiva Using SPLV-entrapped Antibiotic

11.3. Treatment of Keratoconjunctivitis Resulting From SubcutaneousInfections

11.4. Evaluation of the Effectiveness of SPLVs as Compared to LiposomePreparations in the Treatment of Ocular Infections

12. Example: Treatment of Viral Infections

12.1. Treatment of Lethal Lymphocytic Chorio-Meningitis Virus Infectionsin Mice

13. Example: Enhancement of Antibacterial Activity in TreatingCorynebacterium Renale Pyelonephritis Using SPLVs Containing Gentamicinand Nafcillin

13.1. Preparation of SPLVs

13.2. Infection of Mice Using Corynebacterium Renale

13.3. Treatment of Infected Mice

14. Example: Enhancement of Antibacterial Activity in TreatingPseudomonas Aeruginosa Pyelonephritis Using SPLVs Containing Tobramycinand Ticarcillin

14.1. Treatment of Infected Rats

14.2. Effects of Different Treatments of Infected Rats

15. Example: Enhancement of Antibacterial Activity Against ClostridiumNovyi Using SPLVs Containing Gentamicin and Clindamycin

15.1. Infection of Mice Using Clostridium Novyi

15.2. Treatment of Infected Mice

1. FIELD OF THE INVENTION

This invention relates to liposomes and their uses as carriers. Morespecifically, it relates to a new type of lipid vesicle having uniqueproperties which confer special advantages such as increased stabilityand high entrapment efficiency.

The compositions and methods described herein have a wide range ofapplicability to fields such as carrier systems and targeted deliverysystems. The practice of the present invention is demonstrated herein byway of example for the treatment of brucellosis, the treatment of asystemic Salmonella infection, the treatment of ocular infections, thetreatment of pyelonephritis and the treatment of lymphocytic meningitisvirus infections.

2. BACKGROUND OF THE INVENTION 2.1. Liposomes

Liposomes are completely closed bilayer membranes containing anentrapped aqueous phase. Liposomes may be any variety of unilamellarvesicles (possessing a single membrane bilayer) or multilamellarvesicles (onion-like structures characterized by concentric membranebilayers each separated from the next by a layer of water).

The original liposome preparations of Bangham et al. (1965, J. Mol.Biol. 13:238-252) involved suspending phospholipids in an organicsolvent which was then evaporated to dryness leaving a waxy deposit ofphospholipid on the reaction vessel. Then an appropriate amount ofaqueous phase was added, the mixture was allowed to "swell", and theresulting liposomes which consisted of multilamellar vesicles(hereinafter referred to as MLVs) were dispersed by mechanical means.The structure of the resulting membrane bilayer is such that thehydrophobic (non-polar) "tails" of the lipid orient toward the center ofthe bilayer while the hydrophilic (polar) "heads" orient towards theaqueous phase. This technique provided the basis for the development ofthe small sonicated unilamellar vesicles (hereinafter referred to asSUVs) described by Papahadjapoulos and Miller (1967, Biochim. Biophys.Acta. 135:624-638). These "classical liposomes" (MLVs and SUVs),however, had a number of drawbacks not the least of which was a lowentrapment of aqueous space markers.

Efforts to increase the entrapped volume involved first forming inversemicelles or liposome precursors, i.e., vesicles containing an aqueousphase surrounded by a mono-layer of lipid molecules oriented so that thepolar head groups are directed towards the aqueous phase. Liposomeprecursors are formed by dispersing the aqueous solution to be entrappedin a solution of polar lipid in an organic solvent. The liposomeprecursors are then added to an aqueous medium and evaporated in thepresence of excess lipid. The resultant liposomes, consisting of anaqueous phase entrapped by a single lipid bilayer are dispersed inaqueous phase (see U.S. Pat. No. 4,224,179 issued Sep. 23, 1980 to M.Schneider).

In another attempt to maximize the efficiency of entrapmentPapahadjopoulos (U.S. Pat. No. 4,235,871 issued Nov. 25, 1980) describesa "reverse-phase evaporation process" for making unilamellar andoligolamellar lipid vesicles also known as reverse-phase evaporationvesicles (hereinafter referred to as REVs). According to this procedure,the aqueous material to be entrapped is added to a mixture of polarlipid in an organic solvent. Then a homogeneous water-in-oil type ofemulsion is formed and the organic solvent is evaporated until a gel isformed. The gel is then converted to a suspension by dispersing thegel-like mixture in an aqueous media. The REVs produced consist mostlyof unilamellar vesicles (large unilamellar vesicles or LUVs) and someoligolamellar vesicles which are characterized by only a few concentricbilayers with a large internal aqueous space. Certain permeabilityproperties of REVs were reported to be similar to those of MLVs and SUVs(see Szoka and Papahadjopoulos, 1978, Proc. Natl. Acad. Sci. U.S.A.75:4194-4198).

Liposomes which entrap a variety of compounds can be prepared, however,stability of the liposomes during storage is invariably limited. Thisloss in stability results in leakage of the entrapped aqueous solublecompound from the liposomes into the surrounding media, and can alsoresult in contamination of the liposome contents by permeation ofmaterials from the surrounding media into the liposome itself. As aresult the storage life of traditional liposomes is very limited.Attempts to improve stability involved incorporating into the liposomemembrane certain substances (hereinafter called "stabilizers") whichaffect the physical properties of the lipid bilayers (e.g., steroidgroups). However, many of these substances are relatively expensive andthe production of such liposomes is not cost-effective.

In addition to the storage problems of traditional liposomes a number ofcompounds cannot be incorporated into these vesicles. MLVs can only beprepared under conditions above the phase-transition temperature of thelipid membrane. This precludes the incorporation of heat labilemolecules within liposomes that are composed of phospholipids whichexhibit desirable properties but possess long and highly saturated sidechains.

2.2. Uses of Liposomes

Much has been written regarding the possibilities of using liposomes fordrug delivery systems. In a liposome drug delivery system the medicamentis entrapped during liposome formation and then administered to thepatient to be treated. Typical of such disclosures are U.S. Pat. No.3,993,754 issued on Nov. 23, 1976, to Rahman and Cerny, and U.S. Pat.No. 4,145,410 issued on Mar. 20, 1979, to Sears, U.S. Pat. No. 4,235,871issued Nov. 25, 1980, to Papahadjopoulos and Szoka and U.S. Pat. No.4,224,179, issued Sep. 23, 1980 to Schneider.

Desirable features of drug delivery systems depend upon the conditionbeing treated. For example, when treating conditions which requiremaintenance doses of medication, resistance to rapid clearance of thedrug accompanied by a sustained release of the drug which will prolongthe drug's action increases the effectiveness of the drug and allows theuse of fewer administrations. However, if one is treating anintracellular infection, the maintenance of stability in biologicalfluids, until the point that the liposome is internalized by theinfected cell, is critical as is release of the liposome entrapped drugin its bio-active form. Some of the problems encountered in usingliposome preparations in vivo include the following:

(1) Liposome-entrapped materials leak when the liposomes are in contactwith body fluids. This has been attributed to the removal of theliposomal phospholipids by plasma lipoproteins, or to the degradation ofthe liposome membrane by phospholipases, among other reasons. A resultof the degradation of the liposomes in vivo is that almost all theliposomal contents are released in a short period of time, therefore,sustained release and resistance of the drug to clearance are notachieved.

(2) On the other hand, to effect a time release of the entrappedmaterial, if a very stable liposome is used in vivo (i.e., liposomeswhich do not leak when in contact with body fluids in vivo or in vitro),then the liposomal contents will not be released as needed. As a result,these stable liposomes are ineffective as time-release carriers oftherapeutic substances in vivo because the sustained release or theability to release the liposomal contents when necessary is notaccomplished.

(3) Liposomes are internalized by the phagocytic cells of thereticuloendothelial system (RES), and, therefore, are cleared from thesystem rapidly, rendering the entrapped drug largely ineffective againstdiseases involving cells other than the RES. On the other hand, becausecells of the RES phagocytose liposomes, liposome entrapped drugs may bevery useful in treating intracellular infections of the RES. However,after phagocytosis, the liposomal contents are packaged within lysosomesof the phagocytic cell and very often the degradative enzymes containedwithin the lysosome will degrade the entrapped compound or render thecompound inactive by altering its structure or modifying the compound atits active site.

(4) The liposome carriers normally used in delivery systems areexpensive and production is not cost-effective. For example, an improvedmethod for the chemotherapy of leishmanial infections using liposomeencapsulated anti-leishmanial drugs has been reported by Steck andAlving in U.S. Pat. No. 4,186,183 issued on Jan. 29, 1980. The liposomesused in the chemotherapy contained a number of stabilizers whichincreased the stability of the liposomes in vivo. However, as previouslymentioned, these stabilizers are expensive and the production ofliposomes containing these stabilizers is not cost-effective.

(5) Ultimately, the problem encountered in the use of liposomes ascarriers in drug delivery systems is the inability to effect a cure ofthe disease being treated. In addition to rapid clearance anddegradation of the entrapped compound, a number of other explanationsfor the inability to cure diseases are possible. For instance, theliposomes may not deliver a dose which is effective due to the lowpercentage of entrapment of active compound into the vesicles whenprepared.

3. SUMMARY OF THE INVENTION

This invention presents a new and substantially improved type of lipidvesicles which hereinafter will be referred to as stable plurilamellarvesicles (SPLVs). Aside from being structurally different fromconventional liposomes (i.e., MLVs, SUVs and REVs or LUVs), SPLVs arealso prepared differently, possess unique properties, have verydifferent pharmacological and pharmacokinetic effects, and present avariety of different advantages when compared to conventional liposomes.As a result of these differences, SPLVs overcome many of the problemspresented by conventional lipid vesicles heretofore available.

A heterogeneous mixture of plurilamellar lipid vesicles is realized whenSPLVs are synthesized. Evidence indicates that the lipids in the SPLVsare organized in a novel supramolecular structure. Many of the lipidvesicles possess a high number of bilayers, occasionally in excess ofone hundred.

Contrary to current assumptions regarding the distribution of soluteswithin MLVs, experimental evidence presented herein demonstrates thatthere is an uneven distribution of solutes in the vesicle compartmentssuch that an osmotic gradient exists in MLVs (i.e., solute is depletedin some of the compartments of the MLV). This gradient causes a stresson the bilayers which creates what we call a compressed vesicle. Thiscompression and stress may be responsible for many of the problemsassociated with MLVs such as leakage of entrapped compounds, instabilityduring storage, ineffectiveness when used to deliver entrapped compoundsin vivo, etc. In complete contrast to MLVs, the SPLVs of the presentinvention are characterized by lipid bilayers enclosing aqueouscompartments containing one or more entrapped solutes the concentrationof such solutes in each aqueous compartment being substantially equal tothe concentration of solute used to prepare the SPLV. As a result theSPLVs are not under an appreciable osmotic stress and, therefore, arenot compressed.

The properties of SPLVs include: (1) a concentration of entrapped solutein each of the aqueous compartments of SPLVs which is substantiallyequal to the concentration of solute used to prepare the SPLVs; (2)substantially non-compressed bilayers; (3) the entrapment of materialsat a high efficiency; (4) X-ray diffraction signatures that differ fromthat of MLVs; (5) the ability to cure certain diseases which otherdrug-vesicle combinations cannot cure; (6) greatly increased stabilityof the SPLVs during storage in buffer; (7) the increased ability ofSPLVs to withstand harsh physiologic environments; (8) the ability tostick to tissues and cells for prolonged periods of time; (9) theability to release entrapped material slowly in body fluids; (10) thedelivery and ultimate dispersal of the liposomal contents throughout thecytosol of the target cell; (11) the release of compounds in theirbioactive forms in vivo and (12) improved cost-effectiveness inpreparation.

Due to the unique properties of SPLVs they are particularly useful ascarriers in delivery systems in vivo. Methods for the use of SPLVs forthe delivery of bioactive compounds in vivo and the treatment ofpathologies, such as infections, are described.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 graphically demonstrates the difference in membrane stability (asreflected by % leakage) between MLVs and SPLVs treated with varyingconcentrations of urea.

FIG. 2 The ratio of captured volume (μl|μmole) to moles of lipid used isshown for (a) MLVs and (b) SPLVs. For MLVs, this ratio varies littleover the range of lipid used. For SPLVs, the ratio rises sharply forsmall amounts of lipid. In both case, the solute was sodium ⁵¹ chromate;unentrapped material was removed by centrifugation.

FIG. 3 graphically demonstrates the difference in the ability ofascorbate to reduce doxyl spin probes in SPLVs and in MLVs.

FIG. 4 represents the X-ray long spacing versus temperature for MLVs andSPLVs composed of egg phosphatidylcholine. The characteristic forms ofthe MLV and SPLV curves constitute the Long Spacing Signature, or LSS(see text). The LSS curve for SPLVs prepared by the emulsificationprocess is the same as the LSS curve for SPLVs prepared by themonophasic solvent system process.

FIG. 5 represents the small-angle X-ray diffraction intensity (arbitraryunits) versus inverse reciprocal distance for the following liposomescomposed of egg phosphatidylcholine: (a) MLVs; (b) SPLVs prepared by theemulsification process; and (c) SPLVs prepared by the monophasic solventsystem process. The first and second orders on either side of the beamstop shadow (dip near the center) are relatively sharp. However, thesecond order MLV diffraction (n=2) is wider than for SPLVs and oftenasymmetric. Horizontal bars indicate the full width at half maximum forthe first and second order diffraction. This is the second X-raysignature (the Bragg Peak Signature) which distinguishes MLVs fromSPLVs. T=40° C.

FIG. 6 The Wide-Angle X-ray diffraction Signature is shown for thefollowing liposomes composed of egg phosphatidylcholine (a) SPLVsprepared by the emulsification process and MLVs; and (b) SPLVs preparedby the monophasic solvent system process. The signatures of MLVs andSPLVs differ as shown, however, this X-ray signature is more variablethan the LSS. The low-angle camera geometry is unsuitable fordiffraction for s⁻¹ less than 3.7 Å T=10° C.

FIG. 7 The LSS for DOPC liposomes. In this and subsequent figures of theLSS, data points were acquired at 40° C., 20° C., and 0° C. (in thatorder). These data points are connected by straight lines to guide theeye. However, it is important to realize that the true shape of the SPLVcurve is smooth, as seen in FIG. 4.

FIG. 8 The LSS for SPLVs composed of egg phosphatidylcholine and fornegatively charged MLVs. Negatively charged MLVs were prepared using eggphosphatidylcholine and phosphatidic acid in a molar ratio of 8:2.

FIG. 9 The effect of preparing egg phosphatidylcholine liposomes in0.145M salt but suspending them in 0.29M salt is to make the LSS curveof SPLVs look like the LSS curve of MLVs; however both curves differ asto the absolute values.

FIG. 10 The effect of preparing egg phosphatidylcholine MLVs in 0.145Msalt but suspending them in 0.073M salt is to make the LSS look similarto the LSS of SPLVs.

FIG. 11 Egg phosphatidylcholine MLVs which are prepared in 0.435M salt,but suspended in 0.145M salt yield an SPLV-like LSS. This is another wayof enforcing a concentration gradient of the same sign as FIG. 12.

FIG. 12 Nystatin in the presence of cholestrol is known to be anon-specific ionophore. MLVs were prepared using eggphosphatidylcholine, 6.6 mole % cholesterol and 1 mole % nystatin. TheMLV LSS is shown after a few hours and after 24 hours. Note that the MLVLSS relaxes to the LSS of the SPLV control. The MLV and SPLV controlscontained cholesterol but no nystatin. The LSS of these controls did notchange over the 24 hours period. As seen in the MLV control, cholesterolaffects the LSS but at low concentrations, the effects are small.

FIG. 13 SPLVs and MLVs which were prepared using egg phosphatidylcholineand suspended in a sucrose solution without any salt also yield thefamiliar distinct LSS for each type of liposome.

FIG. 14. The LSS for egg phosphatidylcholine liposomes prepared andsuspended in distilled water. The two curves demonstrate an SPLV-likeLSS.

FIG. 15 The ratio of release of ¹⁴ C aqueous marker counts (N_(C)) to ³H membrane headgroup counts (N_(H)) is shown as a function of time asthe liposomes are enzymatically digested. The steep rise of the MLVcurve suggests that the outer aqueous compartments of the MLVs have arelatively low concentration of the aqueous marker solute molecules. At120 minutes, each of the liposomes tested had released about 30% of thetotal headgroup counts. The MLV curve has been scaled by the factor(N_(C) /N_(H))_(SPLV) TOT. x (N_(H) /N_(C))_(MLV) TOT. to allow a directcomparison of the two curves.

FIG. 16 graphically represents the retention of both the lipid andaqueous phases of SPLVs in the eyelid tissues of mice, and the sustainedrelease of ¹²⁵ I-gentamicin from the SPLVs in vivo.

FIG. 17 represents the pharmacokinetics of gentamicin entrapped in MLVsor SPLVs administered intraperitoneally in mice. Antibiotic activityretrieved from the liver (a) and spleen (b) are plotted versus time indays.

FIG. 18 graphically represents the effectiveness of a two stagetreatment of Brucella canis infections in mice using SPLV-entrappedstreptomycin based on B. canis recoverable from spleens of infectedmice.

FIG. 19 graphically represents the effectiveness of a two stagetreatment of B. canis infections in mice using SPLV-entrappedstreptomycin based on B. canis recoverable from organs of infected mice.

FIG. 20 graphically represents the effectiveness of a two stagetreatment of Brucella abortus in guinea pigs using SPLV-entrappedstreptomycin.

5. DETAILED DESCRIPTION OF THE INVENTION

SPLVs are lipid vesicles, the lipid bilayers of which are characterizedby a supramolecular organization which differs from that of conventionalliposomes. Many of the lipid vesicles possess a high number of bilayers,occasionally in excess of one hundred. The membrane bilayer is composedofa bimolecular layer of an amphipathic lipid in which the non-polarhydrophobic hydrocarbon "tails" point inward towards the center of thebilayer and the polar, hydrophilic "heads" point towards the aqueousphase. Occluded by the bilayers is an aqueous compartment, part of whichmakes up the lumen of the vesicle, and part of which lies betweenadjacentlayers. Unlike other multilamellar lipid vesicles, theconcentration of solutes entrapped in each of the aqueous compartmentsof SPLVs are substantially equal to the concentration of solute used toprepare the SPLVs and the bilayers are substantially non-compressed.Complexed with the lipid bilayers can be a variety of proteins,glycoproteins, glycolipids, mucopolysaccharides, and any otherhydrophobic and/or amphipathic substance.

5.1. Preparation of SPLVS

SPLVs may be prepared by any process that results in a substantiallyequal concentration of entrapped solutes in each aqueous compartment ofa plurilamellar lipid vesicle that is substantially equal to theconcentration of solutes used to prepare the SPLVs. SPLVs can beadvantageously prepared by the processes described below which result inaliposome product unique from any other liposome previously described.

5.1.1. Monophasic Solvent System Process

Monophasic solvent system process: a lipid or a mixture of lipids and anaqueous component are added to an organic solvent or a combination oforganic solvents in amounts sufficient to form a monophase. The solventorsolvents are evaporated until a film forms. Evaporation may beaccomplishedby various methods, including but not limited to vacuum(e.g., by rotoevaporation or by passing an inert gas (e.g., nitrogen)over the monophase. Then an appropriate amount of aqueous component isadded, and the film is resuspended and agitated in order to form theSPLVs.

The organic solvent or combination of solvents used in the process mustbe miscible with water and once mixed with water should solubilize thelipidsused to make the SPLVs.

For example, an organic solvent or mixture of solvents which satisfiesthe following criteria may be used in the process: (1) 5 ml of theorganic solvent forms a monophase with 0.2 ml of aqueous component and(2) the lipid or mixture of lipids is soluble in the monophase.

Solvents which may be used in this process of the present inventionincludebut are not limited to ethanol, acetone, 2-propanol, methanol,tetrahydrofuran, glyme, dioxane, pyridine, diglyme,1-methyl-2-pyrrolidone, butanol-2, butanol-1, isoamyl alcohol,isopropanol, 2-methoxyethanol, or a combination of chloroform:methanol(e.g., in a 1:1 ratio).

According to the present embodiment of the process of the invention theevaporation should be accomplished at suitable temperatures andpressures which maintain the monophase and facilitate the evaporation ofthe solvents. In fact, the temperatures and pressures chosen are notdependentupon the phase-transition temperature of the lipid used to formthe SPLVs. The advantage of this latter point is that heat labileproducts which havedesirable properties can be incorporated in SPLVsprepared from pbospholipids such as distearoylphosphatidycholine, whichcan be formed into conventional liposomes only at temperatures above thephase-transition temperature of the phospholipids. This process usuallyallows more than 30-40% of the available water-soluble material to beentrapped during evaporation and 2-15% of the available water-solublematerial to be entrapped during resuspension; and up to 70-80% of theavailable lipid-soluble material can be entrapped if the lipid:drugratio is increased significantly. With MLVs the entrapment of theaqueous phase,which only occurs during the rehydration step since noaqueous phase is present during the drying step, usually does not exceed10%.

The following is an illustrative example of the proportions that may beused in SPLV synthesis using the monophasic solvent system process:SPLVs may be formed by adding 127 micromoles of phospholipid to 5 ml ofethanol and then adding 0.2 ml of aqueous component containing theactive substance to be encapsulated. The resultant solution whichcomprises the material to be entrapped and the entrapping lipid issonicated (sonicationis an optional step) while streaming an inert gasover the mixture, thus removing most of the solvent and forming a film.To the resulting film is added 5-10 ml of aqueous component. Theresuspended film is agitated in order to produce SPLVs. In order toentrap one or more agents in SPLVs, the agent or agents may be added tothe monophase prior to evaporation andformation of the film.Alternatively, the agent or agents may be added withthe aqueouscomponent used to resuspend the film and form the SPLVs. In fact, toobtain a high entrapment efficiency, the agent or agents may be added toboth the monophase and to the aqueous component used to resuspendthefilm. Two or more agents can also be entrapped in one SPLV preparationby adding one agent to the monophase and the other to the aqueouscomponent used to resuspend the film. See U.S. application Ser. No.633,481, filed Jul. 25, 1984 and U.S. application Ser. No. 518,912,filed Aug. 1, 1983 which are incorporated by reference herein.

5 1.2. Emulsification Process

Emulsification process: An amphipathic lipid or mixture of lipids isdissolved in an organic solvent. Many organic solvents are suitable, butdiethyl ether, methylene chloride, fluorinated hydrocarbons and mixturesof fluorinated hydrocarbons and ether are preferred. To this solutionare added an aqueous phase and the active ingredient to be entrapped.This biphasic mixture is converted to SPLVs by emulsifying the aqueousmaterialwithin the solvent while evaporating the solvent, using anyevaporative technique, e.g., evaporation by passing a stream of inertgas over the mixture, by heating, or by vacuum. The volume of solventused must exceed the aqueous volume by a sufficient amount so that theaqueous material canbe completely emulsified in the mixture. Inpractice, a minimum of roughly 3 volumes of solvent to 1 volume ofaqueous phase may be used. In fact theratio of solvent to aqueous phasecan vary to up to 100 or more volumes of solvent to 1 volume aqueousphase. The amount of lipid must be sufficient so as to exceed thatamount needed to coat the emulsion droplets (about 40mg of lipid per mlof aqueous phase). The upper boundary is limited only bythe practicalityof cost-effectiveness, but SPLVs can be made with 15 gm oflipid per mlof aqueous phase.

According to this embodiment of the present invention, the entireprocess can be performed at a temperature range of 4°-60° C. regardlessof the phase transition temperature of the lipid used. The advantage ofthis latter point is that heat labile products which have desirableproperties, for example, easily denatured proteins, can be incorporatedin SPLVs prepared from phospholipid such asdistearoylphosphatidylcholine, but can be formed into conventionalliposomes only at temperatures above their phase-transition-temperature.The process usually allows more than 20% of the available water solublematerial to be encapsulated and more than 40% of the available lipidsoluble material to be encapsulated. With MLVs the entrapment of aqueousphase usually does not exceed 10%.

The following is an example of the proportions that may be used in SPLVsynthesis using the emulsification process: SPLVs may be formed byadding 50 micromoles of phospholipid to 5 ml of diethyl ether containing5 micrograms of BHT (butylatedhydroxytoluene) and then adding 0.3 ml ofaqueous phase containing the active substance to be encapsulated. Theresultant solution which comprises the material to be entrapped and theentrapping lipid is sonicated while streaming an inert gas over themixture thus removing most of the solvent. This embodiment producesparticularly stable SPLVs partially because of the incorporation of BHTinto the vesicles.

See also Lenk et al., 1982, Eur. J. Biochem. 121:475-482 which describesa process for making liposome-encapsulated antibodies by sonicating andevaporating a solution of cholesterol and phosphatidylcholine in amixtureof chloroform and ether with aqueous phase added, but does notset forth the relative proportions of lipid to aqueous phase.

5.1.3. SPLV Constituents

Most amphipathic lipids may be constituents of SPLVs. Suitablehydrophilic groups include but are not limited to: phosphato,carboxylic, sulphato andamino groups. Suitable hydrophobic groupsinclude but are not limited to: saturated and unsaturated aliphatichydrocarbon groups and aliphatic hydrocarbon groups substituted by atleast one aromatic and/or cycloaliphatic group. The preferredamphipathic compounds are phospholipids and closely related chemicalstructures. Examples of these include but are not limited to: lecithin,phosphatidylethanolamine, lysolecithin, lysophatidylethanolamine,phosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin,phosphatidic acid and the cerebrosides, ether lipids and phytanols.

Specific examples of suitable lipids useful in the production of SPLVsare phospholipids which include the natural lecithins (e.g., egglecithin or soybean lecithin) and synthetic lecithins, such as saturatedsynthetic lecithins (e.g., dimyristoylphosphatidylcholine, ordipalmitoylphosphatidylcholine or distearoylphosphatidylcholine) andunsaturated synthetic lecithins (e.g., dioloyl-phosphatidylcholine ordilinoloylphosphatidylcholine. The SPLV bilayers can contain a steroidcomponent such as cholesterol, coprostanol, cholestanol, cholestane andthe like. When using compounds with acidic hydrophilic groups(phosphato, sulfato, etc.) the obtained SPLVs will be anionic; withbasic groups such as amino, cationic liposomes will be obtained; andwith polyethylenoxy or glycol groups neutral liposomes will be obtained.The size of the SPLVs varies widely. The range extends from about 100 nmto about 10,000 nm (10 microns) and usually about 100 nm to about 1500nm.

Virtually any bioactive compound can be entrapped within a SPLV(entrapped is defined as entrapment within the aqueous compartment orwithin the membrane bilayer). Such compounds include but are not limitedto nucleic acids, polynucleotides, antibacterial compounds, antiviralcompounds, antifungal compounds, anti-parasitic compounds, tumoricidalcompounds, proteins, toxins, enzymes, hormones, neurotransmitters,glycoproteins, immunoglobulins, immuno-modulators, dyes, radiolabels,radio-opaque compounds, fluorescent compounds, polysaccharides, cellreceptor binding molecules, anti-inflammatories, antiglaucomic agents,mydriatic compounds,local anesthetics, etc.

Also suitable for entrapment are combinations of incompatible drugs.Concurrent therapy with certain anti-microbial agents can be complicatedbecause some agents which are particularly effective when used togetherinvitro cannot be formulated in a single mixture at therapeuticconcentrationfor use in vivo due to a number of constraints. Forexample, mixtures of gentamicin and nafcillin at therapeuticconcentrations result in the formation of complexes that precipitate outof solution, and therefore, are not administered in vivo simultaneously.In fact, certain drug combinations are not recommended for use in vivodue to drug incompatibility (i.e., either inactivation of the drug orformation of a precipitate). For example, it has been recommended thatthe following antibiotics not be mixed with any other drug: gentamicin,kanamycin, lincomycin, cephalothin, and ampicillin (Davis and Abbitt,1977, JAVMA 170(2): 204-207). Moreover, certain agents cannot besolubilized in the same medium due to chemical restraints (e.g., a lipidsoluble compound anda water soluble compound). These limitations reducethe possible combinations of agents that may be used to obtainenhancement of biological activity in combined thereapy. For a review ofthe topic see Goodman and Gilman, 1980, The Pharmacological Basis ofTherapeutics Sixth Edition, pp. 1080-1106 and Davis et al., 1980,Microbiology, pp. 574-583. However, as seen from Examples, infra,incompatible drugs (i.e., nafcillinand gentamicin) can be combined inSPLVs to yield concurrent therapeutic results.

5.2. Characterization of SPLVS

SPLVs possess many and occasionally over one hundred bilayers, and assuch are clearly distinct in their properties from liposomes with asingle or few lamellae (e.g., SUVs and LUVs or REVs). Freeze-fractureelectron microscopy indicates that SPLV preparations are substantiallyfree of SUVs, LUVs or REVs. They are, however, similar to MLVs byelectron microscopic techniques although many of their physicalproperties and biological efficacies are different. Thus, the followingdetailed comparison is focused on distinguishing SPLVs from MLVs.

In the instant specification that follows, we have compared SPLVs withclassical MLVs as to biological efficacy. At the same dose ofantibiotic, SPLVs are far more effective than MLVs in treating anobligate intracellular bacterial infection (brucella), and a systemicinfection (salmonella). Part of the explanation of these surprisingfindings may liein the differences in the pharmacokinetics of drugsadministered via the two types of liposomes: whereas MLV- deliveredantibiotic is dramatically reduced in the spleen in about 4 days, theSPLV-delivered antibiotic is still present in high concentrations up to5 weeks later. These remarkableresults immediately prompted two lines ofinquiry: 1) How do SPLVs and MLVsphysically differ? and 2) How does thebody recognize and react to the physical differences?

The idealized multilamellar liposome consists of a sequence ofconcentric lipid bilayer shells enclosing some central aqueous volume.For egg phosphatidylcholine (EPC) liposomes the bilayers are separatedby aqueous layers of a well defined width (approximately 20 Å thick)which is a compromise between the repulsive and attractive forcesbetween the bilayers (Rand, 1981 Ann. Rev. Biophys. Bioeng. 10:277-314.Because liposomes are often intended to be used in vivo as drugcarriers, the aqueous fluid normally contains buffered physiologicalsaline, as well as other dissolved solutes. The standard method forproducing MLVs involves vacuum drying lipid dissolved in a solvent, suchas chloroform, to a thin film onto the bottom of a round-bottom flask.MLVs are formed by adding the aqueous solution and shaking or vortexinguntil the dry film is removed from the wall of the flask.

The exact details whereby the dry lipid film converts to MLVs is notwell understood. Lipid bilayers are semipermeable membranes: waterpasses freely through them but solutes, such as salts, are retained(Bangham et. al., 1967 Chem. Phys. Lipids 1:225). It has been generallyassumed that the aqueous solute concentration is uniform throughout theMLV. In the instant specification we demonstrate that in contrast tothis general assumption, standard methods of producing MLVs result invesicles with aqueous compartments which are depleted in solutes, i.e.,that the MLV formation effects a separation of water from its dissolvedsolutes. This leads to an osmotic stress resulting in a compressedliposome.

By contrast, the SPLV processes disclosed in the present inventionproduce multilamellar liposomes characterized by a concentration ofentrapped solutes in each of the aqueous compartments which issubstantially equal to the concentration of solute used to prepare theSPLVs, relatively little osmotic gradient on the liposomes and,therefore, an uncompressed liposome. As explained infra, the state ofosmotic stress on the liposomesaffects a broad spectrum of physicalproperties of the vesicles and has profound implications on theunderstanding and use of lipid bilayers.

The subsections which follow report on the physical differences betweenSPLVs and MLVs which were prepared using the same ingredients. Themethod of preparation of the SPLVs and MLVs used in these studies isdescribed below:

MLV Preparation

EPC (100 mg in chloroform, Sigma Chemical Co., type VIIe) was rotaryevaporated at room temperature to a thin dry film in a 50 ml roundbottom flask. Occasionally, as described in the text, other lipidsand/or solvents were used. Two ml of an aqueous phase (typically, HEPESbuffer consisting of 72.5 mM KCl, 72.5 mM NaCl, 10 mM HEPES, pH 7.4) wasadded tothe flask. If a particular solute was to be entrapped, it wasalso mixed into the aqueous phase added to the flask. The flask wasvortexed until the lipid film coating the flask was completelysuspended. The suspension was then set on the bench to equilibrate for 2hours after which it was washed four times. Each wash was done by mixingthe suspension with bufferto a total volume of 20 ml, followed bycentrifugation (10,000×g) to pellet the liposomes. The supernatant wasremoved and the pellet was then resuspended for further washes or to adesignated final volume.

SPLV preparation using the emulsification process

EPC in chloroform (100 mg), or, occasionally, other lipids as describedin this text, were rotary evaporated to dryness in 50 ml of round bottomflask. The lipid film was dissolved in 5 ml of ethyl ether. Then, 0.3 mlof the aqueous phase (typically HEPES buffer) was added to theether-lipidsolution. If a particular solute was to be entrapped it wasdissolved in the aqueous phase prior to adding it to the ether-lipidsolution. The two-phase mixture (aqueous and ether) was emulsified in abath sonicator (Laboratory Supplies Co., model G1125P1G) during whichtime a gentle stream of nitrogen was passed over the mixture. This wascontinued for approximately 2 minutes until the ether was largelyevaporated and ether could no longer be smelled. The resulting cake wasresuspended in 10 ml ofbuffer, by swirling the fluid in the flask. Thisliposome suspension was pelleted and washed, as described for MLVs.

SPLV preparation using the monophasic solvent system process

An EPC film was prepared in a 50 ml round bottom flask by rotaryevaporation from chloroform, as for SPLVS. Five ml of 95% ethanol and0.2 ml of HEPES buffer were added to the flask and the flask wasvortexed until the lipid film was dissolved. The result was a monophasesolution oflipid, ethanol, and the buffer. The monophase was then rotaryevaporated todryness. The film contained the solutes which weredissolved in the buffer.This dry film was suspended and washed asdescribed above in the MLV procedure.

5.2.1. Stability of SPLVS in Storage

Stability of a lipid vesicle refers to the ability of the vesicle tosequester its occluded space from the external environment over a longperiod of time. For a lipid vesicle to be useful it is paramount that itbe stable in storage and handling. It will be seen that SPLVs made fromnatural lecithin demonstrate increased stability during storage inbuffer when compared to MLVs made from the same ingredients.

There are two factors that cause vesicles to leak. One is auto-oxidationofthe lipids whereby the hydrocarbon chains form peroxides whichdestabilize the bilayers. This oxidation can be drastically slowed downby the addition of antioxidants such as butylated hydroxy toluene (BHT)to the vesicle preparation. Vesicles can also leak because agents in theexteriorenvironment disrupt the bilayer organization of the lipids suchthat the lipids remain intact, but the membrane develops a pore.

Preparations of lipid vesicles are white in color when first made. Uponauto-oxidation, the preparation becomes discolored (brownish). Acomparison of MLVs to SPLVs prepared using the same lipid and aqueouscomponents reveals that MLVs discolor within one to two weeks whereasSPLVs remain white for at least two months. This is supported by thinlayer chromatography of the constituent lipids which showed degradationofthe lipids in the MLVs but not of the lipids of the SPLVs. When thesevesicles are prepared by adding BHT as well as the other constituents,then MLVs appear slightly discolored within one month whereas the SPLVsremain white and appear stable for at least 6 months and longer.

SPLVs and MLVs are also distinguished by the way they leak moleculesentrapped in the aqueous compartments. In general, SPLVs maintain theirentrapment dramatically longer than MLVs. Evidence indicates that SPLVsare able to sequester an encapsulated agent from molecules as small ascalcium ions for more than six months. Arsenazo III is a dye whichchangescolor from red to blue with the slightest amount of divalentcation present. By encapsulating the dye in SPLVs and adding calciumchloride to the storage buffer it is possible to measure the stabilityof the vesiclesby looking for a color change. This was demonstrated bymixing the calcium sensitive dye Arsenazo III (3 mM) in the buffer usedto prepare the liposomes. The liposomes were then washed to remove anyunentrapped Arsenazo III, suspended in HEPES buffer with 500 mM CaCl₂,purged with N₂, sealed in screw top vials and stored at roomtemperature. Leakage of the Arsenazo III can be readily detected by eyeby the color change with occurs when the dye contacts Ca⁺⁺. Whereas MLVsleaked within a few days, the SPLVs have not leaked after 15 months at4° C.

A number of antibiotics have also been tested and the results alsodemonstrated the stability of SPLV entrapment. When placed in a buffercontaining isotonic saline at neutral pH, SPLVs containing antibioticare stable for more than four months, as demonstrated in Table I. Thesedata indicate that none of the antibiotic originally encapsulated withinthe SPLVs leaked out in the period of the experiment.

                  TABLE I                                                         ______________________________________                                        STABILITY OF EGG PHOSPHATIDYLCHOLINE SPLVS                                    AFTER STORAGE IN SEALED CONTAINERS AT 4° C.                            FOR 41/2 MONTHS.sup.a                                                                    Initial    Leakage    Bioavailability                              Entrapped  Entrapment Into       of Entrapped                                 Drug       %          Supernatant.sup.b                                                                        Drug (%)                                     ______________________________________                                        Streptomycin                                                                             34.1       0          97                                           Sulfate                                                                       Spectinomycin                                                                            37.2       0          84                                           Chloramphenicol                                                                          35.2       0          89                                           Oxytetracycline                                                                          18.8       0          91                                           ______________________________________                                         .sup.a SPLVs were prepared using 127 μM egg phosphatidylcholine (EPC)      and 25 μM drug. At the end of 41/2 months storage at 4° C. the      SPLVs were separated from storage buffer by centrifugation. Serial            dilutions of the SPLV contents and the supernatant were applied to            bacterial lawns in order to determine bioactivity as compared to standard     dilutions of antibiotic.                                                      .sup.b 0 indicates below detectable levels as determined by bioactivity. 

In the following experiments vesicles were prepared which containedradioactive tracer molecules within the occluded aqueous compartments.When placed in a buffer containing isotonic saline at neutral pH, SPLVscontaining antibiotic exhibit prolonged stability in storage. Thevesicleswere prepared, each containing one of the followingradio-labeled drugs: ¹²⁵ I-p-hydroxypropionic acid-derived gentamicinsulfate, ¹⁴ C-indomethacin, and ³ H-inulin. After storage at varioustemperaturesfor 14 days the vesicles were separated from the medium bycentrifugation, and the relative amount of radioactivity that escapedfrom the vesicles into the medium was determined. The resultsdemonstrated that SPLVs were more stable during storage than were MLVs.

These experiments demonstrate that SPLVs are sufficiently stable towithstand storage and handling problems. Although it is possible to makeMLVs which are stable for this long, they must be made from syntheticlipids such as DSPC and thus become prohibitively expensive and are notuseful in many in vivo applications.

5.2.2. Stability of SPLVS in other Environments

Placing lipid vesicles in a medium which contains membrane perturbingagents is a way to probe different molecular organizations. Depending onhow the membrane is organized, different vesicles will responddifferentlyto such agents.

In the following experiments vesicles were prepared which contained aradioactive tracer molecule (³ H-inulin) within the occluded aqueouscompartment. Inulin, a polysaccharide, partitions into the aqueousphase, and thus when radiolabeled may be used to trace the aqueouscontents of lipid vesicles. After an appropriate interval of exposure toa given agent, the vesicles were separated from the medium bycentrifugation, and the relative amount of radioactivity that escapedfrom the vesicles into the medium was determined. These results arereported in Table II; values are expressed as percent leaked, meaningthe proportion of radioactive material in the surrounding mediumrelative to the starting amount encapsulated in the vesicles.

SPLVs are more stable than MLVs in hydrochloric acid. Table IIillustrates that both MLVs and SPLVs, when made from egg lecithin, aredestabilized when exposed to 0.125N hydrochloric acid for one hour.However, it is noteworthy that the SPLVs are considerably lesssusceptible to the acid than MLVs. Presumably this different responsereflects an intrinsic difference in the way the lipids interact withtheir environment.

                  TABLE II                                                        ______________________________________                                        STABILITY OF SPLVS IN OTHER ENVIRONMENTS                                                      % LEAKAGE                                                     Incubating Medium.sup.a                                                                         MLVs    SPLVs                                               ______________________________________                                        Hydrochloric Acid                                                             0.125M            90.5    55.2                                                Urea                                                                          1M                21.7    44.8                                                Guanidine                                                                     0.5M               5.7     7.4                                                1.0M               8.3    10.1                                                Ammonium Acetate                                                              0.5M              27.0    67.0                                                1.0M              25.9    54.7-63.1                                           Serum             76.2    57.8                                                ______________________________________                                         .sup.a Incubation time is 2 to 4 hours except incubation in HCl was for 1     hour at room temperature.                                                

SPLVs also respond differently than MLVs when exposed to urea (FIG. 1and Table II). Urea is a molecule with both a chaotropic effect(disrupts the structure of water) and a strong dipole moment. It isobserved that SPLVs are far more susceptible to urea than they are to anosmotic agent such assodium chloride at the same concentration (FIG. 1).MLVs do not leak significantly more in urea than they would in sodiumchloride. Although the explanations for this different behavior aretheoretical, it would appear that the response is due to the dipoleeffect, rather than a chaotropic property, since guanidine, a moleculesimilar to urea, does notdestabilize SPLVs (Table II). Althoughguanidine is also strongly chaotropic, it does not possess a strongdipole moment.

SPLVs are also susceptible to ammonium acetate, while MLVs are not(Table II). However, neither ammonium ion (in ammonium chloride) noracetate (in sodium acetate) are particularly effective in causing SPLVsto destabilize. Thus it would appear that it is not the ion itself, butthe polarity of the ammonium acetate which is responsible for inducingleakage.

Initially these results seem surprising because SPLVs are more stablethan MLVs when incubated in body fluids such as sera or blood. However atheoretical explanation for these results can be proposed (of courseotherexplanations are possible). If the stability of the SPLV is due tothe unique structure of its uncompressed or unstressed membrane bilayerssuch that the polar groups of the membrane lipids are hydrated by acloud of oriented water molecules, or hydration shell, then it ispossible that anyagent which disrupts or interferes with such hydrationshells would promotechanges in structural membrane integrity, andtherefore, leakage.

Independent of the theoretical explanations for the destabilization ofSPLVs in urea the results serve to demonstrate characteristicdifferences between the structure of MLVs and SPLVs. This differenceserves a very useful purpose in application. As described infra, SPLVsbecome slowly leaky when applied to the eye. Presumably this desiredslow release of contents is due to a similar destabilization of theSPLVs when exposed to tear fluid.

SPLVs are more stable in serum than MLVs. Many applications of lipidvesicles include administering them intraperitoneally, such as for thetreatment of brucellosis. To be effective, the vesicles must survive forasufficient time to reach their desired target. SPLVs and MLVs, bothmade from egg lecithin, were exposed to fetal bovine serum whichcontained active complement, (Table II). After 48 hours exposure at 37°C., SPLVs are demonstrably more stable than MLVs.

5 2.3. Entrapment of Active Material by SPLVS

One measure which distinguishes between liposomes is the efficiency withwhich an aqueous solute may be entrapped. Entrapment efficiency (orencapsulation efficiency) is defined as the fraction of the aqueouscompartment sequestered by bilayers.

As a prime example of the superiority of SPLVs over traditional MLVs,SPLVsentrap a larger percentage of the available active material therebyconserving material (see Table III). Entrapment is measured herein asthe fraction of initial solute remaining with the liposomes after fourwashes.

                  TABLE III                                                       ______________________________________                                        COMPARISON OF MLVS AND SPLVS                                                             % Available Material Entrapped.sup.a                                                                Monophasic                                                        Emulsification                                                                            Solvent System                               Encapsulation of:                                                                          MLVs    Method      Method.sup.b                                 ______________________________________                                        inulin (aqueous                                                                            2-6     20-37       37                                           space marker)                                                                 bovine serum 15      20-50       .sup. ND.sup.c                               albumin                                                                       streptomycin 12-15   20-40       ND                                           polyvinylpyrrolidone                                                                       5       25-35       ND                                           (aqueous space)                                                               .sup.125 I-Gentamicin                                                                      ND      33          38                                           .sup.14 C-Indomethacin                                                                     ND      22          15                                           ______________________________________                                         .sup.a Values are expressed as percent entrapped meaning the proportion o    radioactive material in the liposome pellet (cpm) relative to the starting     amount (cpm) added to the preparation.                                        .sup.b Radiolabeled material to be entrapped was added to the monophase.      After evaporation to a film and resuspension with aqueous buffer to form      SPLVs, the preparation was pelleted and the radioactivity of the              supernatant was determined.                                                   .sup.c Not determined.                                                   

5.2.4. Effect of Varying the Initial Lipid to Aqueous Ratio

Another parameter used to characterize liposomes, called capturedvolume, is defined as the volume enclosed by a given amount of lipid andis expressed as units of liters entrapped per mole of total lipid (1 mol⁻¹). To gain insight as to the formation kinetics we examined thedependence of the processes for preparing MLVs and SPLVs on the initialamount of lipid used. The ratio of solute entrapped to the amount oflipidused in preparing the liposomes (i.e. captured volume) versus thestarting amount lipid was determined by entrapping trace quantities ofradioactively labeled water soluble markers. The amounts of lipid usedto prepare the liposomes were varied from 15 to 658μ moles. Sodium⁵¹chromate (⁵¹ Cr) was entrapped in MLVs and SPLVs which were washed bycentrifugation and then placed in a gamma counter to determine theamount of entrapped ⁵¹ Cr.

As shown in FIG. 2, the ratio of solute entrapped to lipid used wasessentially constant for MLVs. The simplest interpretation of thisresult is that the use of more lipid resulted in the formation of moreMLVs without changing the entrapped ⁵¹ Cr to lipid ratio of individualliposomes. By contrast, SPLVs entrapped more efficiently (FIG. 2) athigh aqueous to lipid ratios, suggesting that the SPLV compositionchanged as the initial lipid concentration was increased.

5.2.5. Volume of SPLVS

Furthermore, when collected in a pellet by centrifugation from 1,000 to100,000×g, SPLVs form a pellet that is substantially larger than MLVs,given the same phospholipid concentration. At a force of 16,000×g, theSPLVs form a pellet approximately one third larger than MLVs.

As shown in Table III (supra) if ¹⁴ C-inulin is dissolved in thewaterused to prepare the liposomes, MLVs typically entrapped 2% of heinulin whereas SPLVs entrapped 37%. If one assumes that 2%×2 ml=40 μlofthe inulin solution is entrapped in MLVs and that 37% of 0.3 ml=111 ulis entrapped in SPLVs then the latter should contain 111/40=2.8 times asmuchfluid. In fact, upon centrifugation, SPLVs yielded pellets whichwere larger than MLVs, but only by about 30%. Consequently, thedifference in entrapment efficiency cannot be simply due to thedifference in the volumeof the initially entrapped aqueous fluid. Whenthe experiment was done with ²² NaCl, the MLVs entrapped 2% and theSPLVs entrapped 27%.

MLV and SPLV pellets behaved quite differently. After centrifugation(10,000×g), and upon pouring off the supernatant, the MLV pelletremained intact in the bottom of the test tube. By contrast, the SPLVpellet was soft and tended to run out of the tube with the last of thesupernatant. This difference was apparant even after the firstpreparativewash.

5.2.6. Buoyant Density of SPLVS

Additionally, SPLVs have a lower buoyant density than MLVs. This ismeasured by banding in a ficol gradient in which SPLVs layer above 0.5%ficol whereas MLVs layer above 1% ficol.

5.2.7. Osmotic Properties of SPLVS

Since phospholipid bilayers are permeable to water, placing liposomes ina hypertonic environment drives the occluded water out due to osmoticforce.When placed in a hypertonic environment, SPLVs shrink more thanMLVs. In addition, after shrinking 16 hours in a buffer that is twentytimes higherthan the internal salt concentration, SPLVs do not shrink tothe same finalvolume as MLVs (SPLV pellets remain 1/3 larger than MLVpellets). This indicates that the difference in pellet size is not dueto differences in aqueous enclosed volume.

In fact, the osmotic properties of MLVs greatly differ from those ofSPLVs.The unequal concentrations of solute in the aqueous compartmentsof the MLVand the solute depletion in its outer layers creates anosmotic gradient that compresses the MLV. In contrast, the concentrationof solute in each aqueous compartment of the SPLVs which issubstantially equal to the concentration of solute used to prepare theSPLVs results in uncompressed lipid vesicles. This is discussed in moredetail infra.

5.2.8. Electron Spin Resonance

Although SPLVs and MLVs appear similar by electron microscopy, ESR(electron spin resonance) spectroscopy reveals differences in theirsupramolecular structure. SPLVs can be distinguished from MLVs on thebasis of their molecular architecture as evidence by greaterpenetrabilityto ascorbate. It is possible that these differences inmolecular architecture contribute to their different biological effects.

In electron spin resonance spectroscopy a spin probe such as 5-doxylstearate (5DS) is incorporated into the lipid bilayer. The unpairedelectron of the doxyl group absorbs microwave energy when the sample isinserted into a magnetic field. Both SPLVs and MLVs were labeled with5-doxyl stearate as follows: SPLVs and MLVs were made as previouslydescribed, except that 1 mole percent of 5-doxyl stearate (MolecularProbes, Junction City, Oreg.) was added to 40 mg of EPC in chloroformprior to the initial rotary evaporation step. After the formation ofliposomes, the preparations were washed again and ESR spectra of bothsamples were recorded.

An illustration of the differences between SPLVs and MLVs resides in theability of ascorbate to reduce doxyl spin probes. It has been known forsome time that ascorbate reduces doxyl moieties presumably to theirhydroxylamine analogs which do not absorb microwave energy in a magneticfield. In aqueous solutions the reduction occurs rapidly withconcomitant loss of ESR signal. If the spin probe is in a protectedenvironment such as a lipid bilayer it may be reduced more slowly or notat all by the hydrophilic ascorbate. Thus the rate of nitroxidereduction can be used tostudy the rate of penetration of the ascorbateinto lipid bilayers. FIG. 3 shows the percentage remaining spin versustime for SPLVs and MLVs suspended in an ascorbate solution. Followingthe addition of ascorbate (10 mM final concentration), spectra wererecorded at regular intervals inorder to follow reduction of signal as afunction of time. At 90 minutes the ascorbate has reduced 25% of theprobe embedded in MLVs but 60% of theprobe embedded in SPLVs. Thesimplest interpretation is that SPLVs allow for a dramatically greaterpenetrability of ascorbate than do MLVs.

5.2.9. X-Ray Diffraction

X-ray diffraction was applied in an attempt to define signatures whichcould be used to distinguish MLVs from SPLVs. Three different such X-raydiffraction signatures were found and are referred to herein as the LongSpacing Signature (LSS), the Bragg Peak Signature and the Wide AngleX-raySignature.

5 2 9.1. X-Ray Diffraction Methods

X-rays were generated on a Rigaku RU-200 X-ray generator using a 0.2×2mm focus cup and a loading of 50 KV, 60 mA. The beam was focussedhorizontally via single-mirror Franks optics and collimated verticallyas described in Gruner, 1977, The application of an efficient X-raydetector to diffraction from retinal rod outer segment membranes, Ph.D.Thesis, Princeton University, Princeton, N.J. X-rays were detected usinga quantum-limited 2-dimensional slow-scan TV detector (Gruner, ibid.;Reynolds et al., 1978, Rev. Sci. Instr. 49:1241-1249), yielding theX-rayintensity in each of 240×240 adjacent areas or pixels. Typical X-rayexposure times were 5-30 seconds.

The small angle diffraction consisted of concentric rings of Braggorders arising from the liposome multilayer repeat spacing. X-raypatterns were real-time reduced to 1-dimensional traces of intensity vs.scattering angle by radial integrations over 20°-50° of the2-dimensional pattern. Multilayer repeat spacings were determined by aleast-square fit to the peak positions of the Bragg orders, where thepeakpositions were taken as the centers of parabolas least-squares fitto the peak profiles.

Wide-angle X-ray patterns were acquired via the TV-detector and reducedto 1-dimensional traces via radial integration. As opposed to thesmall-anglepatterns the high -angle integrations at each radius weredivided by the length of the arc integrated at that radius.

A typical X-ray run consisted of diffraction patterns taken at severaltemperatures with 2 minute equilibration times after temperaturechanges.

5.2.9.2. X-Ray Diffraction Signatures

In the small-angle regime, both SPLVs and MLVs exhibit three lamellarorders of diffraction which arise from the radial stacking of membranesinthe liposome. If the X-ray repeat spacing (defined as the sum of thethicknesses of a lipid bilayer and of the inter-bilayer aqueous space)is graphed versus temperature, the curves for MLVs and SPLVs made usingphosphatidylcholine (or any zwitterionic lipid) were seen to differ incharacteristic ways (FIG. 4):

a) MLVs exhibited a repeat spacing which fell linearly with increasingtemperature. By contrast, the SPLV curve flattened out at a highertemperatures.

b) The MLV curve fell below the SPLV curve.

c) The two curves approached one another at 0° C.

These three characteristics constitute the first of the diffractionsignatures, hereinafter referred to as the Long Spacing Signature, orLSS.The LSS is the most reliable and easily quantified of the threeX-ray signatures to be discussed: for a given type of liposomalpreparation the curves are repeatable to within 0.5 Å. Variation of theliposomal parameters (e.g., lipid or buffer composition) may cause theabsolute values of the curves to shift but, in so far as the liposomesare phenomenologically distinguishable as SPLVs and MLVs, the LSSappears to distinguish between the two types of liposomes. Furthermore,the area detector that was used can generally acquire the neededsmall-angle X-ray exposures in 5-30 seconds. Moreover, the thermalkinetics of the LSS were at least as fast as our ability to slew overtemperature (0.3° C./S) and acquire the data. Consequently, it was anexperimentally convenient signature in that it could be acquired in afew minutes.

A second signature (herein referred to as the Bragg Peak Signature)which distinguished MLVs from SPLVs was the width and asymmetry of theBragg peaks (FIG. 5a, b & c). When interpreting Bragg peaks ofliposomes, one should compare the full width at half maximum of thepeaks obtained for each order (this is indicated by horizontal bars inFIG. 5). The difference between the full width at half maximum of thefirst and second order peaks is greater for MLVs than for SPLVs.Furthermore, MLVs (FIG. 5a) exhibit peaks which are broader and oftenasymmetric. This may readilybe interpreted as arising from a largerstatistical variation of repeat spacings in the MLVs. The angle ofdiffraction, 2θ, for a given order, n, follows from the Bragg relation

    nλ=2D sin θ,                                  (Eq.1)

where

λ=X-ray wavelength and

D=repeat spacing of the lattice.

Suppose the sample contains a distribution of repeat spacingscharacterizedby a width, D. Then, for sinθ≃θ, the spread in scatteringangle is simply ##EQU1##Note that |Δθ| increases with the order number,n. This is why the peak asymmetry in FIG. 5a is only apparent in thesecond order peak (n=2); the asymmetry of the first order peak is hiddenby the instrumental line width due to the X-ray camera. The use of thisX-ray signature requires careful measurement of the instrumentallinewidth which, in practice, is harder to measure than the repeatspacing. This signature also is more variable than the LSS. For thesereasons, the LSS is the preferred signature to aid in identificationpurposes.

The third X-ray signature was in the wide-angle regime (λ/2θ<10 Å) andis referred to herein as the Wide-angle X-ray Signature. Melted-chainlipids exhibit a broad peak at about 4.4 to 4.6 Å due to correlations inthe hydrocarbon region (Luzzati, 1968, X-ray diffraction studes of lipidwater systems; in Biological Membranes, Vol. 1., D. Chapman, ed.,Academic Press, N.Y.; Costello & Gulik-Krywicki,1976 Biochim. Biophys.Acta 445:412-434). As shown in FIG. 6 the MLVs yielded a well-definedpeak at 4.4-4.6 Å but SPLVs exhibit diffractionwhich extends to muchhigher angles. This indicates that SPLVs have electron-densitycorrelations which vary over a wider range of distances; in particular,over distances smaller than 4 Å. This signature requires a rearrangementof the X-ray detector geometry, tended to vary, and is harder toquantify than the LSS.

Of the three signatures, the width and asymmetry of the small-anglediffracted orders (i.e., the Bragg Peak Signature shown in FIG. 5) ismosteasily understood. For liposomes consisting of many layers, theshape of the diffracted orders is a direct reflection of the width andasymmetry ofthe distribution of membrane repeat spacings. (N.B., UnlikeSPLVs, unilamellar and oligolamellar vesicles will not demonstrate aBragg Peak Signature because there is no respective lattice which can bedetected by X-ray diffraction.) MLVs exhibit relatively wide, asymmetricpeaks indicative of a repeat spacing whose distribution mode and meandiffer. This is consistent with the data of FIG. 5a since a non-uniformdistribution of solutes would lead to non-uniform osmotic forces betweenthe layers and result in a slight variation in the repeat spacings.

The LSS (FIG. 4) is relatively insensitive to the repeat spacingdistribution; rather, by definition, it maps out the variation in themeanrepeat spacing as the temperature is varied. Since the repeatspacing is the sum of the thicknesses of the bilayer and of theinter-bilayer aqueousspace, both these component widths may be expectedto be thermally sensitive. As the temperature is raised, more gaucherotamers are excited in the bilayer hydrocarbon, resulting in a thinnermembrane and an increase in the area per molecule (Reiss-Husson, 1967,J. Mol. Biol. 25:363). In the presence of excess water, the thickness ofthe fluid spaceis set by a complicated balance of Van der Waals,hydration, membrane tension, and osmotic forces. In particular, thestrong hydration force is likely to be coupled to the area/molecule,since it is known that the area/molecule changes as membranes arerehydrated (Small, 1967, J. Lipid Res. 8: 551-557). In addition,although there has been little investigation of the thermal variation inthe hydration force at a fixed area/lipid molecule, it would besurprising if the force coefficients werethermally insensitive. Thus,there is a complicated interaction between thethicknesses of the waterand lipid layers such that forces which act laterally in the lipid planealso affect both the lipid and water thicknesses. Our understanding ofthe statistical mechanics of the hydrocarbon, of the interactions in thelipid polar region, and of the hydration force is not yet sufficientlysophisticated to completely predict the LSS. (N.B., because the LSS mapsthe mean repeat spacing against temperature, the LSS is directly relatedto the Bragg peak signature. If there are no peaks in the Braggsignature, no LSS can be obtained. Therefore, unlike SPLVs, unilamellarand oligolamellar vesicles will not demonstrate an LSS.)

The Wide-angle X-ray Signature (FIG. 6) is also poorly understood.Broad, diffuse diffraction in the 4.6 to 3.5 Å range arises primarilyfrom density correlations in the lipid hydrocarbon, although we cannotexclude the possibility that the wide-angle SPLV signal arises, in part,from water associated with the bilayers. The wide-angle signature may beexpected to be less sensitive to changes in the aqueous region than theLSS.

5.2.9.3. Long Spacing Signature

The text below describes the use of the LSS to examine the liposomeswhich resulted when the normal procedures for preparing MLVs and SPLVswere altered.

It was first hypothesized that the differences between the signatures ofMLVS and SPLVs may have resulted from contamination by the solvents usedin the liposome preparations. When the liposomes were made, bothchloroform and ethyl ether were used. These solvents, like manyanesthetics, alter membrane properties (Janoff & Miller, 1982, acritical assessment of the lipid theories of general anesthetic action,in Biological membranes, Vol. IV., D. Chapman, ed., Academic Press,London). To address whether residual ether contamination was important,SPLV ether was measured via gas chromatography using a Beckman GC72-Egas chromagraphequipped with a flame ionization dectector and a SpectraPhysics "minigrator" electronic integrator. The column was a WatersAssociates copper 6:×1/4" poropak P. The carrier flow was 60 cc/min. Thedetection limit was 250 μM.

After 1 wash, 13 mole % ether remained; after the 2nd wash, 0.5 mole %was left; after the 3rd wash, the remaining ether was below thedetectable limit of 0.1 mole %. Even so, ether was added to thechloroform-lipid stock solution used to prepare MLVs to see if thisaffected the MLVs. The resulting liposomes were indistinguishable fromMLVs made without ether asindicated by the LSS (data not shown).Consequently, it was concluded that ether cannot account for thedifferences between the signatures obtained for MLVs and SPLVs.

Even though both MLVs and SPLVs were prepared from a lipid film driedfrom chloroform, it is possible that SPLVs would not contain the sameconcentration of residual chloroform because residual chloroform mayhave evaporated with the ether. To test if residual chloroform wasinvolved, the lipid films used to produce MLVs and SPLVs were vacuumpumped for 72 hours and then washed 8 times in double the normal volumeof wash buffer. The resulting LSS for each was indistinguishable fromthat of normal MLVs and SPLVs. We conclude that chloroform cannot beresponsible for the differences between the signatures obtained for theliposomes.

Another hypothesis was that the SPLV process produced bilayers whichdiffered in acyl chain composition in the inner and outer leaflets. Thiswas conceivable because EPC is a mixture of lipids and contains aspectrumof acyl chains. To test this hypothesis, the liposomes were madeout of pure DOPC (dioleoylphosphatidylcholine, Avanti Polar Lipids,Birmingham, All), which contained only oleoyl chains. The LSS (FIG. 9)demonstrated DOPC MLVs and SPLVs to be distinct, thereby ruling outmembrane asymmetry.

To determine whether the LSS of MLVs can be made to look like that ofSPLVssimply by putting a charge on the membrane, MLVs composed of eggphosphatidylcholine and phosphatidic acid in a molar ratio of 8:2 weremade and examined by X-ray diffraction. The results shown in FIG. 8 showthat negatively charged MLVs do not have the same LSS as SPLVs.

5.2.9.4. Relation of Long Spacing Signature to the Osmotic Properties ofSPLVS and MLVS

Besides lipids and water, the remaining major constituents of liposomeswere the salts dissolved in the buffers used to prepared the vesicles.

When an osmotic stress is imposed upon a multilayered liposome manyaspectsof the structure of the vesicle are affected. As previouslydiscussed the three X-ray signatures each probe different, althoughrelated, parts of the liposome structure. In principle, it is possibleto stress bilayers indifferent ways so as to affect some parts of thestructure more than others. The result is that a change in one of theX-ray signatures need not necessarily be reflected by simple changes inthe others.

Experiments demonstrated that the salt concentration gradient across theliposomes, as opposed to the amount of the entrapped salt, is importantinaffecting the LSS. For example, FIG. 9 shows that if SPLVs wereprepared in0.145M salt (i.e., physiological concentration), butsuspended in 0.290M salt, the resulting LSS resembles that of MLVs. Thusit appeared that a MLV-like LSS resulted if an osmotic gradient wasimposed on SPLVs by increasing the salt concentration outside theliposomes (i.e., by exposingthe SPLVs to a hypertonic environment). TheLSS typical of SPLVs could similarly be achieved by suspending MLVs in asolution of low salt concentration (i.e., by exposing the MLVs to ahypotonic environment). FIG. 10 demonstrates, indeed, that MLVs preparedin 0.145M salt but suspended in 0.073M salt exhibit a SPLV-like LSS. Thefact that the salt gradient is important is also seen in FIG. 11, whichshows the LSS of MLVsprepared with 0.435M salt and then suspended in0.145M salt; the LSS is again seen to be typical of that of that ofSPLVs.

FIGS. 9-11 established that a salt concentration gradient (salt depletedinside the liposome) was sufficient to cause SPLVs to reveal a MLV-likeLSS and that relaxing or reversing this gradient yielded a SPLV-likeLSS. A further test of this hypothesis was to prepare liposomes with anon-specific ionophore, such as nystatin (Andreoli & Monahan, 1968, J.Gen. Physiol. 52:300-325: Holz & Finkelstein, 1970, J. Gen. Physiol. 56:125-145), incorporated into the bilayers. In the presence ofcholesterol, this ionophore allows passage of salt ions and wouldgradually relax a salt gradient. As would be predicted, the LSS of MLVscontaining nystatin gradually converted to the LSS of SPLVs (preparedwithout nystatin) as thesalts leaked through the nystatin pores (FIG.12).

To investigate whether the differences between the liposomes were due toanosmotic gradient or an ionic gradient associated with a salt gradient,liposomes were prepared in 0.290M sucrose, which is a non-ionic soluteanda first approximation to the osmolarity of the 0.145M salts that arenormally used. FIG. 13 shows that the LSS differences between MLVs andSPLVs are preserved, suggesting that an osmotic gradient is sufficient.

The fact that an osmotic gradient is important in affecting the LSS maybe seen in FIG. 14 which shows the LSS of liposomes prepared andsuspended indistilled water. Note that under these conditions, the LSSof both MLVs andSPLVs yielded SPLV-like curves.

It should be noted that a SPLV-like LSS resulted if SPLVs were eitherprepared with no osmotic stress (FIG. 14) or with solutions which weresomewhat more concentrated than the buffer outside the liposome. Thelatter statement follows from the fact that the 0.145M salt used toprepare SPLVs lost about 10% of its water when the ether was evaporatedduring the SPLV formation process, thereby concentrating solutes.

The LSS is subject to variation which depends upon the osmoticconditions of the buffer used in suspending the liposomes. The abilityto adjust conditions such that the LSS of a liposome can be manipulatedto be MLV-like or SPLV-like does not mean that SPLVs and MLVs are thesame. As previously mentioned, the LSS measures only one parameter ofSPLVs. For instance, an MLV placed in a hypotonic solution will swellresulting in a change in its LSS, however the other X-ray signatures maynot be affected.Furthermore, a change in the LSS of the MLV due toswelling of the MLV in ahypotonic solution does not indicate a change inthe entrapment or distribution of solute molecules in the MLV. As isexplained in detail below, in contrast to SPLVs, the MLV remainssolute-depleted regardless ofthe change in its LSS when placed in ahypotonic solution.

5.2.9.5. Solute Distribution in SPLVS vs. MLVS

The hypothesis that MLVs are solute depleted was also tested directlyvia NMR. It is known that the ³¹ P-NMR signal that is associated withphosopholipid is quenched by Mn⁺⁺. If MLVs and SPLVs are preparedandsuspended with a buffer containing Mn⁺⁺, one expects the integrated³¹ P-NMR signal to be less (per unit of lipid) for SPLVs then for MLVs.The reason for this is that the SPLVs should have Mn⁺⁺ entrapped betweenthe bilayers and, thus, accessible to all the ³¹ P;consequently, the ³¹P-NMR signal should be strongly quenched. By contrast, if MLVs aresolute and Mn⁺⁺ depleted, less of the lipid signal should be quenched.

SPLVs and MLVs were prepared and suspended in HEPES buffer containing 2mM MnCl₂. ³¹ P-NMR spectra were collected at 20° C. employing a BrukerWP 200 Fourier transform NMR spectrometer operating at 81 MHz for ³¹ P.Accumulated free induction decays (FID's) from 5,000transients wereobtained using a 20 KHz sweepwidth, a 7μ sec 90° radio frequency pulseand a 1 sec interpulse time in the presence of gatedbroad band protondecoupling. An exponential multiplication corresponding to 50 Hz linebroadening was applied to the FID prior to Fourier transformation.

MLV and SPLV signals were accumulated under identical samples were runimmediately after each other so that the relative signal intensitieswere accurate. The integrated ³¹ P-NMR spectra were normalized by theSPLVand MLV concentrations (84.2 mg/ml and 109.9 mg/ml, resp.) so as tobe directly comparable. The integrated SPLV signal intensity was foundto be 72% that of MLVs; thus, as expected, the SPLV signal was stronglyquenched.

The data thus far presented suggests that SPLVs have a concentration ofsolute in each of the aqueous compartments of the liposomes that issubstantially equal to the concentration of solute used to prepare theSPLV but that MLVs are solute depleted. It is likely during thehydration of the dry lipid film used to make MLVs water gains access tothe space between the bilayers but most of the solutes were excluded. Ifit were possible to deposit a dry lipid film with solutes alreadybetween the layers, then the resultant liposomes might be expected tobehave like SPLVs even though the dry lipid film is hydrated by the MLVprocess. This,in fact, is the monophasic solvent system processdescribed in section 5.1.1. A solute-containing lipid film can beprepared by solubilizing lipid in a solvent (e.g., ethanol) which isalso miscible with water, drying the mixture under vacuum, and thenrehydrating the resulting film in buffer. Note that this final hydrationstep is identical to that used to prepare MLVs; only the lipid film withwhich one starts is different. The LSS of SPLVs prepared by themonophasic solvent system process is similar to that of SPLVs preparedby the emulsification process, as were the other two X-ray signatures(see FIGS. 4-6). This, again, suggested that MLVs are solute depleted.

Information about the distribution of solute within liposomes (i.e., thesolute concentration profile) can be obtained via enzymatic digestion ofthe lipid. To this end liposomes were prepared with a radioactive markerincorporated in the lipid headgroups and a different isotopic markerincorporated in the aqueous solution. The liposomes were slowly degradedby exposure to phospholilpase C, an enzyme which cleaves the lipidheadgroup from the glycerol backbone. The amount of headgroup markerreleased by the enzyme is a measure of the number of lipid moleculesdigested, which should also be proportional to the area of the lipidbilayer involved. As a layer of the liposome breaks down, the aqueousmarker entrapped beneath that layer should also be released.Consequently,the ratio of the amount of headgroup to aqueous markersreleased measures the ratio of bilayer area to aqueous marker entrappedbeneath that layer. The sharp X-ray diffraction peaks indicate that,even for MLVs, the aqueous space between the bilayers is constant towithin a small fraction of an Angstrom. Because all the aqueous layersare essentially of fixed width, the amount of aqueous marker releasedper unit area of membrane is proportional to the aqueous marker soluteconcentration in that layer. Mathematically, if dN_(H) /dt and dN_(A)/dt are the rates of release of the headgroup and aqueous markers,respectively, then

    (dN.sub.A /dt)/(dN.sub.H /dt)=dN.sub.A /dN.sub.H =αc (Eq. 3)

where

c=concentration of aqueous marker and

α=a proportionality constant.

If the aqueous marker concentration varies with depth in the liposome,thenthe ratio of marker release (the left hand side of Eq. 3) will varyover time as the enzyme degrades deeper and deeper layers of theliposomes. If,for example, the aqueous marker concentration between theouter bilayers isof lower concentration than that between the deeperbilayers then one expects the ratio N_(A) /N_(H) to initially rise andthen level out asthe liposomes degrade.

This experiment was performed by incorporating a small amount of ³H-DPPC (³ H-dipalmitoylphosphatidylcholine) as a headgroup markerandentrapping ¹⁴ C-sucrose as an aqueous marker dividing the liposomesuspensions into 9 aliquots and adding Phospholipase C. Periodicallyover the course of the enzyme digestion, aliquots were centrifuged tosettle the liposomes and the supernatant was scintillation counted todetermine the amount of released headgroup and aqueous marker counts.The method is described in detail below:

MLVs and SPLVs were made containing a ³ H-label in the membranes and a¹⁴ C-label in the aqueous spaces by mixing trace quantities ³ H-DPPCwith the EPC and by preparing the liposomes in buffer containing tracesof ¹⁴ C-sucrose. The ³ H-DPPC had the label on the choline head groups.These double labeled liposomes were washed 4 times and divided into nine0.5 ml aliquots so that each aliquot contained 5.0 mg of lipid.Phospholipase C (0.2 units) was added to each fraction and timing wasinitiated. Immediately, and every 15 minutes thereafter for 2 hours, onefraction from each group was removed, centrifuged at 10,000×g and 50 μlof the aqueous supernatant recovered and the counts/minute of each labelwas determined by liquid scintillation counting. Phospholipase C digestsphosphatidylcholine by cleaving the phosphorylcholine head groups whichcontain the ³ H-label. Thus, the two reaction products are ³H-phosphorylcholine and mixed diglycerides. Since neither of thesecompounds are capable of maintaining membrane integrity the ¹⁴ C-sucroseentrapped within the aqueous space circumscribed by a bilayer will bereleased as the bilayer is digested. Upon centrifugation, three distinctlayers are seen: a layer of diglycerides floating on the surface, aclear aqueous supernatant, and a pellet composed of undigestedliposomes. Since both ¹⁴ C-sucrose and ³ H-phosphorylcholine are watersoluble, counting the supernatant layer will indicate the amount ofmembrane digested, as well as the amountof solute (¹⁴ C-sucrose) whichindicates the amount of membrane digested/amount of solute released.

The results are shown in FIG. 15. Note that the relative initial aqueousmarker concentration of the MLVs is very low and then rises slowly whilethe aqueous marker concentration of the SPLVs is much more nearlyconstant. The simplest interpretation of FIG. 15 is that MLVs areespecially solute depleted in the outer layers while SPLVs are ofessentially uniform concentration throughout. Note that thisinterpretation is dependent on assumptions of sequential degradation ofthe liposome layers and is also complicated by heterogeneity (size,numberof layers, etc.) in the liposomes. We note in passing that bothMLVs and SPLVs were digested at roughly comparable rates.

5.2.9.6. Identification of SPLVS

In view of the foregoing results, the following criteria may be used toidentify an SPLV:

1) the lipid vesicle should be multilamellar and therefore have ademonstrable LSS and peaks in its Bragg Signature;

2) the lipid vesicle should have a high entrapment efficiency;

3) the lipid vesicle should be characterized by a concentration ofsolute in each aqueous compartment that is substantially equal to theconcentration of solute used to prepare the SPLVs;

4) the lipid vesicle should be characterized by substantiallynon-compressed bilayers;

5) the LSS of a vesicle composed of zwitterionic lipids should besubstantially as depicted for SPLVs in FIG. 4, provided the vesicles aresuspended in a buffer that is isosmotic to the aqueous medium which wasused to prepare the vesicles;

6) the Bragg Peak Signature of a vesicle composed of zwitterionic lipidsshould be substantially as depicted for SPLVs in FIG. 5, provided thevesicles are suspended in a buffer that is isosmotic to the aqueousmediumwhich was used to prepare the vesicles; and

7) the Wide-angle X-ray Signature of a vesicle composed of zwitterioniclipids should be substantially as depicted for SPLVs in FIG. 6, providedthe vesicles are suspended in a buffer that is isosmotic to the aqueousmedium which was used to prepare the vesicles.

5.2.9.7. Interpretation of Results

Although not wishing to be limited or bound to any model or theory ofoperation of the present invention, we believe it will be helpful ininterpreting the data to offer a possible theoretical explanation whichcan be summarized by the following model that may explain thedifferences between SPLVs and MLVs: the critical physical differencebetween SPLVs andother multilamellar lipid vesicles seems to be thatSPLVs are characterizedby a concentration of entrapped solute in eachaqueous compartment that is substantially equal to the concentration ofsolute used to prepare the SPLVs and that unlike MLVs, SPLVs do not havean osmotic stress and, therefore, the bilayers are not undercompression.

Knowledge of the internal composition of MLVs has been hampered by alack of detailed information about how a dry lipid film reacts whenexposed to solute-containing water. The prevailing view point has beensummarized by Bangham et al. (1974, Meth. Membr. Biol. 1:1-68), who havewritten in a review about liposomes that "their usefulness as a modelsystem derived from the fact that, as the dry phospho- and/or otherlipids of biological origin undergo their sequence of molecularrearrangments, there is an opportunity for unrestricted (emphasis added)entry of solutes, e.g., isotopically labeled salts and proteins, betweenthe planes of hydrophilicheadgroups before an unfavorable entropysituation of an oil-water interface intervenes." They go on to say thatthere is a subsequent sealing of membranes into concentric, closedcompartments, sequestering water and solutes which can thereafter onlydiffuse across compartments bycrossing the bilayer walls. It wasrealized shortly after the development of the MLV procedure by Banghamand colleagues (1965, J. Mol. Biol. 13: 238-252), that lipid bilayersare highly permeable to water and relativelyimpermeable to alkali metalsalts and a host of other solutes (Bangham et al., 1967, Chem. Phys.Lipids 1:225). This being the case, one may ask if the sealing of theoutermost bilayer wall, upon hydration of a dry film, occurs prior to,or after the enclosed lipid has been fully hydrated. If the sealing wereto occur before full hydration, then water will continue to enter, viadiffusion, because lipid headgroups have a high natural affinity forwater (Parsegian et al., 1979, Proc. Natl, Acad. Sci. U.S.A.76:2750-2754). However, salts and other such solutes may be excludedbecause of the relative impermeability of the outermost bilayer wall tothese substances. The result would be a liposome in which the internalconcentration of solute in the aqueous layers would be less than in theexternal bulk water. However, we know of no place in the literaturewhere the question of the last four sentences is posed and discussed. Wealso know of no study which has examined the distribution of solutewithin liposomes formed by the MLV procedure. In the absence of suchdiscussion it has generally been implicitly assumed that the aqueoussolute concentration inside the liposomes is the same as theconcentration of thefluid in which the liposomes are formed. Forexample, in an elegant study of the osmotic and swelling properties ofliposomes, Bangham et al. (1967 supra) prepared liposomes in 50 mM KCl."Aliquots (0.5 ml) were then pipetted into test tubes containing 1.5 mlof 250, 117, 50 and 17 mM tracer-free KCl and 1.5 ml water,respectively. These solutions represented osmotic stresses calculated toquarter, halve, leave unalteredand quadruple the osmotically activespaces inside . . ." The quantities involved reveal the authors to beassuming that the initial inner concentration is 50 mM, the same as thesolution in which the liposomes were prepared.

In attempting to understand the differences between SPLVs and MLVs, oneis forced to question if the internal MLV solute concentration is thesame asthat of the fluid used to prepare the liposomes. The high soluteentrapmentefficiency of SPLVs relative to MLVs raised the questions ofwhere the entrapped solute was being held within the liposome. In thepast, the low entrapment efficiency of MLVs seemed reasonable in view ofan inadequate knowledge of specific adsorption by the bilayers, and thewide heterogeneity in the number of bilayers and sizes of the centralaqueous volumes the liposomes. However, when MLVs and SPLVs werecompared, these reasons were rapidly seen to be inadequate to explainthe differences in entrapment efficiency. Electron microscopy offreeze-fractured liposomes and histogram analysis revealed similar sizedistribution. Specific adsorption could not account for the differencesbecause both types of liposomes were made of the same lipid. X-raydiffraction revealed the repeat spaces to differ by only about 2 Å (25°C.). Using an established bilayer thickness of roughly 40 Å (Small,1967, J. Lipid Res. 8: 551-557; Worcester, 1976, Neutron beam studies ofBiological Membranes and membrane components; in biological membranes,vol. 3., Chapman and Wallach, eds., Academic Press.), this implied thatthe aqueousthickness between the bilayers differed by only 10-15%. Largedifferences in the central aqueous volume or in the number of layerscould not be reconciled with the fact that the SPLV and MLV pelletvolumes per unit mass of lipid were comparable to within 30%. Itappeared that MLVs were somehow diluting the solutes or SPLVs wereconcentrating them.

The X-ray signatures described herein provided a rapid means ofevaluating hypotheses as to the differences between the two types ofliposomes. It was seen that an X-ray diffraction signaturecharacteristic of MLVs could be obtained from SPLVS by increasing theexternal salt concentration and vice versa for MLVs by decreasing theexternal salt concentration relativeto the concentration of the solutionused to prepare the liposomes. This suggested that the X-ray signatureswere sensitive to a concentration gradient. The LSS was reproduced withsucrose suggesting that the different X-ray signatures were primarilydue to osmotic, as opposed to ionic, effects. Although changing the saltcomposition has strong effects on the LSS, MLVs and SPLVs still alwaysdiffer. For example, the entrapment efficiency of MLVs is lower thanthat of SPLVs: this parameter would not change despite the change in theLSS. When liposomes were prepared in distilled water to remove anyconcentration gradients, both MLVs and SPLVs yielded an SPLV-like LSS,suggesting that MLVs had lower internal solute concentration. Theionophore, enzyme digestion and NMR experiments all supported thepicture of an MLV which is under osmotic compression.

We have already discussed how the early sealing of a bilayer skin whichexcludes solute may lead to low solute concentrations in MLVs. Bycontrast, we envision SPLVs are resulting from the dynamics ofcoalescenceof inverted water-in-ether droplets in which the lipid actsas a surfactant.

Importance of Osmotic Stress

The liposome literature is large. There are so many different ways toprepare liposomes, and so many lipids from which to prepare them, thatit is fair to ask: what variables are important? Obviously, the answerto this question depends on the use which is intended for the liposomes.However, one of the most often stated reasons for studying liposomes isfor the use as an in vivo drug carrier. We were alerted to thedifferencesbetween MLVs and SPLVs by their vastly differentpharmacokinetics. SPLVs are highly effective in curing infectiousdiseases, whereas MLVs are not. SPLVs are also far more stable thanMLVs. Given these important differences, let us now consider thevariables whose investigation forms the vast bulk of the liposomeliterature.

Literally speaking, both SPLVs and MLVs are multilamellar. Much of theliterature is concerned with the differences between multilamellar andunilamellar liposomes. These differences are important but do notdifferentiate MLVs from SPLVs. One of the reasons given for usingunilamellar liposomes is that they can have a high entrapment efficiency(e.g., Szoka and Papahadjopoulos, 1978, Proc. Natl. Acad. Sci. U.S.A.79: 4194-4198). While this is true, in light of the results presentedherein, this is not a compelling argument against the use ofmultilamellar vesicles, since SPLVs also have a high entrapmentefficiency.

Another highly discussed variable is the size of the liposomes. Again,thisis important but does not differentiate MLVs from SPLVs. Yet anothervariable involves purity of the lipids and reagents. We certainly wouldnever imply that this is an unimportant variable. However, the studiesdescribed herein were done with EPC straight from the bottle, withoutadditional purification steps; solvents were of reagent grade and usedwithout additional purification. Exposure to air was minimized but by nomeans eliminated. Yet the reproducibility of the distinctions betweenMLVsand SPLVs has been excellent. Again, we do not mean to belittleproblems ofcontamination. We only wish to emphasize that this variableis secondary with respect to the structural distinctions between SPLVsand MLVs. It cannot for instance, account for the storage stabilitydifferences betweenthe two types of liposomes.

The lipid composition of liposomes is the concern of an enormousliterature. But both MLVs and SPLVs can be made of EPC. Review of theexperiments discussed Section 5.2 will show that, within narrow limits,the lipid composition can be varied without erasing the distinctionbetween MLVs and SPLVs. Neither the buffering system, nor the specificionic composition is of paramount importance. The most importantvariable appears to be the sign and magnitude of the osmotic stressesexerted upon liposomes. Although numerous studies have used the osmoticproperties of liposomes, this osmotic stress has not been recognized asa variable, perhaps the most important variable, with which todifferentiate certain liposomal types.

Why is the osmotic stress important? More fundamentally, what do thedifferences in the X-ray signature tell us? We only know partial answersto these questions. Recent research in osmotically induced fusion (Cohenet al., 1980, J. Gen. Physiol 75:251-270; and 1982 Science 217:458-460;Zimmerberg et al., 1980) has empirically demonstrated the importance ofunderstanding osmotic stress across bilayers. The bulk of the liposomeliterature deals with the chemical properties of bilayers; relativelylittle emphasis has been placed on the material properties of lipidbilayers when stressed in various ways. Consider an MLV under osmoticstress of a sign such as to make the liposome expand. This is the caseforFIG. 10. One sees that the LSS of MLVs under this type of stresslooks likethe LSS of a normal SPLV. The liposome has surely dilated whensuspended inthe hypotonic buffer. In doing so the bilayer shell mustincrease their area. In so far as lipids are constrained to a givenbilayer shell, this means the area per molecule must also increase. Thishas enormous implications because once the area per molecule changesthen the interaction of all the forces in the system must change. In thevarious models of the statistical mechanics of membranes, an orderparameter that has a profound effect is the area per molecule. This hasbeen emphasized, for example by Nagle (1980, Ann. Rev. Phys. Chem.31:157-195), by Israelachvili et al. (1980 Biophys. 13:121-200), by Pink(1982 Theoreticalmodels of phase changes in one- and two- componentlipid bilayers; in Biological Membranes, vol. 4, D. Chapman, ed.,Academic Press, N.Y. pp. 131-178),and by Kirk et al., (1984), Biochem.23:1093-1102). As the area/molecule changes, the hydrocarbon orderparameter must also change. Several studies (see Israelachivili et al.,1980 supra) have examined the hydrocarbon order parameter state ofinclination from the membrane normal)vs. carbon number down the chain.We know of no studies which have done this for stressed bilayers. TheWide-angle X-ray signature (FIG. 6), suggests this order parameterprofile changes under stress. In fact, in most statistical mechanicalmodels, the area/molecule is introduced as a fundamental constraint oras a parameter derived from an independent calculation of the headgroupinteractions. The change in the area/moleculeis likely to affect thehydrogen bonding and steric interactions between head-groups. It mustalso affect the magnitude of the interaction with water: it is knownthat the thickness of a bilayer is coupled to the hydration of themultilameter lattice. The area/molecule decreases as water is withdrawnfrom the lattice (Luzzati, 1968 X-ray diffraction studies of lipid watersystems; in Biological membranes, Vol 1., D. Chapman, ed., AcademicPress, N.Y. pp. 71-123; Rand, 1981 Ann. Rev. Biophys. Bioeng.10:277-314). We suggest the coupling may be reversible, i.e., thatexpanding or shrinking the area/molecule may affect the interaction withwater. In sum, the entire molecular basis of the bilayer is affected bythe osmotic stress. As a result, osmotically compressed liposomes behavedifferently than unstressed liposomes even though both are composed ofthe same species of lipids. Note, incidentally, in FIGS. 9-11 that it isthe direction and magnitude of the osmotic gradient, whichseem mostimportant.

An MLV is a many layered vesicle with one small core. Assume this coreis either iso-osmotic to the suspending buffer or, where bent, is bentinto radii so small that it is constrained against collapse (recall thatunilamellar vesicles have a minimum size of roughly a few hundred Å(Mason & Huang, 1978, Annals N.Y. Acad. Sci. 308: 29-49. Further assume,as we believe is the case for MLVs, that the outer layers are solutedepleted. The osmotic stress is of a direction such as to collapse theouter layers by withdrawal of water. However, as opposed to aunilamellar vesicle, the outer layers cannot collapse because they arewrapped around a "rigid" core. The thickness of the water layers betweenthe bilayers cannot shrink appreciably because, as Parsegian, Rand andcoworkers have shown (see Rand, 1981, Ann. Rev. Biophys Bioeng.10:277-314 for a review) very strong repulsive hydration forces keep thebilayers apart. Parsegian et al. (1979, Proc. Natl. Acad. Sci. U.S.A.76: 2750-2754) have measured the repeat spacing for EPC in pure water at25° C.; they obtain a value of 62.5 Å. Reference to FIG. 4 shows the MLVvalue to be 61.7 Å, indicating that the MLV layers are collapsed againstthe "hard-wall" of the hydration force. (Note, incidentally, that therepeat spacing of either MLVs or SPLVs in pure water, i.e., unstressedbilayers, as shown in FIG. 14, is about 62.5 Å at 25° C.). The outerlayers of the MLV, in fact, are under a stress which has hitherto beenrarely considered in liposomes: the bilayers are under compression. Incomplete contrast, the bilayers of SPLVs are uncompressed.

Solute depletion of the outer and possibly many inner layers of MLVs hasmany other implications. For example, one of the solutes most commonlyinvolved is NaCl. The Nernst equations states that an imbalance of theconcentrations, C¹ and C₂, of an ionic species across a membranegivesrise to an electrical potential

    Δφ=-(RT/zF) ln (c.sub.2 /c.sub.1)                (Eq. 4)

Where

R=the gas constant

T=temperature

z=ionic charge

Δφ=electrical potential and

F=Faraday's number.

Now the bilayer permeability coefficient for Na⁺ (10⁻¹² cm/S) is almost2 orders of magnitude smaller than for C1⁻. This means that the Na⁺gradient cannot relax in weeks; the C1⁻ gradient relaxesalmost 100 timesfaster, leading to a Nernst potential across the bilayer of MLVs. Thebiological implications of this are hard to access but maybesignificant. The potential may be responsible for the rheologicaldifferences between MLV and SPLV pellets.

5.3. Uses of SPLVS

SPLVs are particularly useful in systems where the following factors areimportant: stability during storage and contact with body fluids; arelatively high degree of encapsulation; cost-effectiveness; and thepreservation of the biologically active form of the entrapped compound.

Furthermore, depending upon the mode of administration in vivo, SPLVscan be resistant to rapid clearance (e.g., where sustained delivery isimportant) or can be delivered to the cells of the RES.

As a result, the SPLVs of the invention are usefully employed in a widevariety of systems. They may be used to enhance the therapeutic efficacyof medications, to cure infections, to enhance enzyme replacement, oraldrug delivery, topical drug delivery, for introducing geneticinformation into cells in vitro and in vivo, for the production ofvaccines, for the introduction of recombinant deoxyribonucleic acidsegments into cells, or as diagnostic reagents for clinical testsfollowing release of entrapped "reporter" molecules. The SPLVs can alsobe employed to encapsulate cosmetic preparations, pesticides, compoundsfor sustained slow release toeffect the growth of plants and the like.

The methods which follow, while described in terms of the use of SPLVs,contemplate the use of SPLVs or any other liposome or lipid vesiclehavingfunctional characteristics similar to those of SPLVs.

5.3.1. Delivery of Bioactive Compounds

SPLVs demonstrate a number of characteristics which make themparticularly suitable as carriers for delivery systems in vivo:

(A) When administered by a non-intravenous or non-intraperitoneal route,SPLVs are resistant to clearance. When SPLVs are administered to anorganism by routes including but not limited to subcutaneously,topically,intramuscularly, and the like, both the lipid component andthe entrapped active ingredient are retained in the tissues and by thecells to which they are administered;

(B) SPLVs can be engineered to provide sustained release. The stabilityof SPLVs is "adjustable" in that SPLVs are very stable during storageand arestable in the presence of body fluids but when administered invivo a slow leakage of the active ingredient permits the sustainedrelease of the active ingredient;

(C) Because of the high level of entrapment and stability whenadministered, effective doses of the active ingredient are released; and

(D) The production of SPLVs is very cost effective in that stability ofthevesicles is achieved without incorporating expensive stabilizers intothe bilayers.

5.3.1.1. Delivery in Vitro

Delivery of compounds to cells in vitro (e.g., animal cells, plantcells, protists, etc.) generally requires the addition of the SPLVscontaining the compound to the cells in culture. Still another benefitof SPLVs is that SPLVs interact with cells such that a relatively largeportion of thematerials encapsulated inside the vesicle is dispersedthroughout the cytoplasm of the cells rather than being limited tophagocytic vesicles. When SPLVs are mixed with cells the two appear tocoalesce. By coalescence, SPLVs, unlike MLVs, interact with cells invitro so that all the cells contain at least some of the materialsoriginally entrapped in the SPLVs. This material appears to bedistributed throughout each cell and not limited to just the phagocyticvesicles. This can be demonstrated by incorporating ferritin in theaqueous phase of a SPLV preparation. After coalescence with a cell inculture, ultrastructural analysis revealsthat the ferritin isdistributed throughout the cytosol and is not bound byintracellularmembranes. While this phenomenon can be shown to occur with MLVs agreater quantity of material can be transferred by SPLVs.

5.3.1.2. Delivery in Vivo

SPLVs, however, can also be used to deliver compounds in vivo in animals(including man), plants and protists. Depending upon the purpose ofdelivery, the SPLVs may be administered by a number of routes: in manand animals this includes but is not limited to injection (e.g.,intravenous, intraperitoneal, intramuscular, subcutaneous,intraarticular, intraauricular, intramammary, intraurethrally, etc.),topical application (e.g., on afflicted areas), and by absorptionthrough epithelial or mucocutaneous linings (e.g., ocular epithelia,oral mucosa, rectal and vaginal epithelial linings, the respiratorytract linings, nasopharyngeal mucosa, intestinal mucosa, etc.); inplants and protists this includes butis not limited to directapplication to organism, dispersion in the organism's habitat, additionto the surrounding environment or surroundingwater, etc.

The mode of application may also determine the sites and cells in theorganism to which the compound will be delivered. For instance, deliveryto a specific site of infection may be most easily accomplished bytopicalapplication (if the infection is external). Delivery to thecirculatory system (and hence reticuloendothelial cells), may be mosteasily accomplished by intravenous, intraperitoneal, intramuscular, orsubcutaneous injections.

Since SPLVs allow for a sustained release of the compound, doses whichmay otherwise be toxic to the organism may be utilized in one or moreadministrations to the organism.

The following experiments demonstrate some of these characteristics ofSPLVs when administered topically onto the eyes of test animals. TheSPLVsused in these experiments were prepared as previously describedexcept thatthe lipid bilayer and the active ingredient were eachradiolabeled in orderto trace these components in the eye tissues over aperiod of time.

SPLVs were prepared using 100 mg egg phosphatidylcholine (EPC) and 100mg gentamicin sulfate. The lipid component was radiolabeled by theincorporation of trace amounts of ¹²⁵ I-phosphatidylethanolamine (¹²⁵I-PE) into the bilayers, whereas the active ingredient in the aqueousphase was radiolabeled by the addition of ¹²⁵ I-gentamicin sulfate (¹²⁵¹-GS). The SPLVs were washed with buffer repeatedly in order toeffectively remove unincorporated or unencapsulated materials.

An aliquot of the SPLV preparation was removed and extracted in order toseparate the organic phase from the aqueous phase. The radioactivity ofeach phase was measured in order to determine the initial ratio of ¹²⁵I-PE:¹²⁵ I-GS (cpm (counts per minute) in the lipid phase:cpm in theaqueous phase) which was incorporated into the SPLVs.

The extraction was done as follows: 0.8 ml of 0.4M NaCl (aqueous), 1 mlchloroform, and 2 ml methanol were mixed to form a homogeneous phase.Then4 μl of the radiolabeled SPLVs were added and mixed; as the SPLVcomponents dissolved into the organic phase and into the aqueous phase,the mixture, which was initially turbid, became clear. The phases wereseparated by adding and mixing 1 ml 0.4M NaCl (aqueous) and 1 mlchloroform, which was then centrifuged at 2,800×g for 5 minutes. Analiquot (1 ml) of each phase was removed and the radioactivity (in cpm)was measured. (The initial ratio of ¹²⁵ I-PE:¹²⁵ I-GS was 1.55:1).

Fifteen adult female Swiss Webster mice were anesthetized and restrained(in order to prevent them from wiping their eyes). Equal aliquots (2 μl)of the radiolabeled SPLVs in suspension were topically applied to eacheye. Groups of three animals were then sacrificed at each of thefollowing points: 1, 2, 3, 18, and 24 hours. Nine female Swiss Webstermice (controls) were treated identically except that equal aliquots (2μl) of an aqueous solution of radio-labeled gentamicin sulfate wereapplied topically to each eye. Groups of three control animals weresacrificed at the end of 1, 4, and 8 hours.

Immediately after sacrifice the eyelids of the animals were removed,minced, and extracted (using the procedure previously described) inorder to separate the aqueous components from the lipid components. Theradioactivity of each phase was determined (as well as the total numberofradioactive counts recovered). The radioactivity measured in the lipidphase is an indication of the retention of SPLV lipids by the eyetissue, whereas the radioactivity measured in the aqueous phase is anindication of the retention of gentamicin in the eye tissue. FIG. 16graphically demonstrates the retention of each component in the eyelidtissue (expressed as the percent of the original number of cpm appliedto the eye).

FIG. 16 clearly demonstrates the retention of the SPLV lipid componentin the eyelid tissue over a 24 hour period, and the sustained release ofgentamicin from the SPLVs over a 24 hour period (as reflected by thepercent gentamicin retained in the eyelid tissue during this time). FIG.17 also demonstrates that unencapsulated gentamicin (aqueous gentamicinadministered topically) is rapidly cleared from the eyelid tissue. Forexample, gentamicin in solution (control) was cleared from the eyelidtissue within 4 hours (less than 5% of the gentamicin remained in theeyelid tissue). On the other hand, more than 50% of theSPLV-encapsulated gentamicin was retained by the eyelid tissue in this 4hour period; in fact, at the end of 24 hours more than 15% of theSPLV-encapsulated gentamicin was retained by the eyelid tissue. Thisindicates that approximately 85% of the SPLV-encapsulated gentamicin wasreleased over a 24 hour period whereas 95% of the unencapsulatedgentamicin sulfate was cleared within a 4 hour period.

Table IV compares the ratio of the SPLV lipid phase:aqueous phaseretained in the eyelid tissue at each time point. An increase in thisratio indicates release of gentamicin from the SPLVs.

                  TABLE IV                                                        ______________________________________                                        SUSTAINED RELEASE OF SPLV-ENCAPSULATED                                        GENTAMICIN AFTER TOPICAL APPLICATION IN                                       EYES OF MICE                                                                           Total SPLV Compo-                                                                             Ratio of SPLV Lipid:                                 Time     nents Recovered Aqueous Phase                                        Post-    from Eyelids    Retained In Eyelids                                  Application                                                                            (% Initial Dose)                                                                              (.sup.125 I-PE: .sup.125 I-GS)                       ______________________________________                                        0            100             1.55                                             1    hr      100             2.1                                              3    hr      100             2.82                                             18   hr       94             6.89                                             24   hr         85.1         7.17                                             ______________________________________                                    

The bioactivity of the SPLV-encapsulated gentamicin sulfate which wasretained by the eyelid tissues was also evaluated. Gentamicin sulfatewas recovered from the eyelid tissues by removing an aliquot from theaqueous phase of the eyelid extracts prepared 3 hours after theSPLV-encapsulated gentamicin sulfate was applied to the eye. The aqueousphase was serially diluted and 2 μl aliquots were placed ontoStaphylococcus aureus lawns on agar plates; after 24 hours incubationthe zones of inhibition were measured. The gentamicin sulfate recoveredfrom the eyelid tissue extractsof animals treated with SPLV-encapsulatedgentamicin sulfate fully retainedits bioactivity.

The difference in the behavior of SPLVs and MLVs in vivo can best beseen by examining the pharmacokinetics of drugs entrapped within eachtype of vesicle. We have entrapped gentamicin in MLVs and SPLVs andstudied the pharmacokinetics of that drug within the liver and spleenfollowing intraperitoneal injection in mice (FIG. 17a and b). Theresults demonstrated in FIG. 17a and b demonstrate that these twovesicles are handled in drastically different ways. Actually, in otherexperiments active antibiotic administered in SPLVs has been found inthe spleen 5 weeks after injection. Little antibiotic given in MLVs canbe observed past 7 days.

The sections which follow describe some overall schemes in which SPLVsmay be used and demonstrate but do not limit the scope of the presentinvention.

5.3.2. Treatment of Pathologies

A number of pathological conditions which occur in man, animals andplants may be treated more effectively by encapsulating the appropriatecompound or compounds in SPLVs. These pathologic conditions include butare not limited to infections (intracellular and extracellular), cysts,tumors andtumor cells, allergies, etc.

Many strategies are possible for using SPLVs in the treatment of suchpathologies; a few overall schemes are outlined below which areparticularly useful in that they take advantage of the fact that SPLVswhen administered in vivo are internalized by macrophages.

In one scheme, SPLVs are used to deliver therapeutic agents to sites ofintracellular infections. Certain diseases involve an infection of cellsof the reticuloendothelical system, e.g., brucellosis. Theseintracellularinfections are difficult to cure for a number of reasons:(1) because the infectious organisms reside within the cells of thereticuloendothelial system, they are sequestered from circulatingtherapeutic agents which cannot cross the cell membrane intherapeutically sufficient concentrations, and, therefore, are highlyresistant to treatment; (2) often the administration of toxic levels oftherapeutic agents are required in order to combat such infections; and(3) the treatment has to be completely effective because any residualinfection after treatment canreinfect the host organism or can betransmitted to other hosts.

According to one mode of the present invention, SPLVs containing anappropriate biologically active compound are administered (preferablyintraperitoneally or intravenously) to the host organism or potentialhostorganism (e.g., in animal herds, the uninfected animals as well asinfectedanimals may be treated). Since phagocytic cells internalizeSPLVs, the administration of an SPLV-encapsulated substance that isbiologically active against the infecting organism will result indirecting the bioactive substance to the site of infection. Thus, themethod of the present invention may be used to eliminate infectioncaused by a variety of microorganisms, bacteria, parasites, fungi,mycoplasmas, and viruses, including but not limited to: Brucella spp.,Mycobacterium spp., Salmonella spp., Listeria spp., Francisella spp.,Histoplasma spp., Corynebacterium spp., Coccidiodes spp., Pseudomonasspp., and lymphocytic choriomeningitis virus.

The therapeutic agent selected will depend upon the organism causing theinfection. For instance, bacterial infections may be eliminated byencapsulating an antibiotic or combinations of antibiotics. Theantibioticcan be contained within the aqueous fluid of the SPLV and/orinserted into the vesicle bilayer. Suitable antibiotics include but arenot limited to: penicillin, ampicillin, hetacillin, carbencillin,ticarcillin, nafcillin, tetracycline, tetracycline hydrochloride,oxytetracycline hydrochloride, chlortetracycline hydrochloride,7-chloro-6-dimethyltetracycline, doxycycline monohydrate, methacyclinehydrochloride, minocycline hydrochloride, rolitetracycline,dihydrostreptomycin, streptomycin, gentamicin, kanamycin, tobramycin,neomycin, erythromycin, carbomycin, oleandomycin, troleandomycin,Polymyxin B collistin, cephalothin sodium, cephaloridine, cephaloglycindihydrate, and cephalexin monohydrate.

We have demonstrated the effectiveness of such treatments in curingbrucellosis, salmonellosis and pyelonephritis (see Examples, infra). Bythe procedure of this invention, the effectiveness and duration ofaction are prolonged. It is surprising that this system is effective fortreatinginfections which do not respond to known treatments such asantibiotics entrapped in MLVs. Successful treatment is unexpected sinceany small remaining infections will spread and the infectious cycle willcommence again. We have also demonstrated success in treatinglymphocytic choriomeningitis virus infection.

Of course, the invention is not limited to treatment of intracellularinfections. The SPLVs can be directed to a variety of sites of infectionwhether intracellular or extracellular. For instance, in anotherembodiment of the present invention, macrophages are used to carry anactive agent to the site of a systemic extracellular infection.According to this scheme, SPLVs are used to deliver a therapeuticsubstance to uninfected macrophages by administering the SPLVs in vivo(preferably intraperitoneally or intravenously). The macrophages willcoalesce with the SPLVs and then become "loaded" with the therapeuticsubstance; in general, the macrophages will retain the substance forapproximately 3 to 5 days. Once the "loaded" macrophages reach the siteof infection, the pathogen will be internalized by the macrophages. As aresult, the pathogen will contact the therapeutic substance containedwithin the macrophage, and be destroyed. This embodiment of theinvention is particularly useful in the treatment of pyelonephritis.

If the site of infection or affliction is external or accessible theSPLV-entrapped therapeutic agent can be applied topically. Aparticularly useful application involves the treatment of eyeafflictions. In the case of ocular afflictions, SPLVs containing one ormore appropriate active ingredients may be applied topically to theafflicted eye. A number of organisms cause eye infections in animals andman. Such organisms include but are not limited to: Moraxella spp.,Clostridium spp., Corynebacterium spp., Diplococcus spp., Flavobacteriumspp., Hemophilus spp., Klebsiella spp., Leptospira spp., Mycobacteriumspp., Neisseria spp., Propionibacterium spp., Proteus spp., Pseudomonasspp., Serratia spp., Escherichia spp., Staphylococcus spp.,Streptococcus spp. and bacteria-like organisms including Mycoplasma spp.and Rickettsia spp. These infections are difficult to eliminate usingconventional methods because any residual infection remaining aftertreatment can reinfect through lacrimal secretions. We have demonstratedthe use of SPLVs in curing ocular infections caused by Moraxella bovis(see examples, infra).

Because SPLVs are resistant to clearance and are capable of sustainedrelease of their contents, SPLVs are also useful in the treatment of anyaffliction requiring prolonged contact with the active treatingsubstance.For example, glaucoma is a disorder characterized by a gradualrise in intraocular pressure causing progressive loss of peripheralvision, and, when uncontrolled, loss of central vision and ultimateblindness. Drugs used in the treatment of glaucoma may be appliedtopically as eyedrops. However, in some cases treatment requiresadministering drops every 15 minutes due to the rapid clearing of thedrug from the eye socket. If an affliction such as glaucoma is to betreated by this invention therapeuticsubstances as pilocarpine,Floropryl, physostigmine, carcholin, acetazolamide, ethozolamide,dichlorphenamide, carbachol, demecarium bromide,diisopropylphosphofluoridate, ecothioplate iodide, physostigmine,orneostigmine, etc. can be entrapped within SPLVs which are then appliedto the affected eye.

Other agents which may be encapsulated in SPLVs and applied topicallyinclude but are not limited to: mydriatics (e.g., epinephrine,phenylepinephrine, hydroxy amphetamine, ephedrine, homatropine,scopolamine, cyclopentolate, tropicamide, encatropine, etc.); localanesthetics; antiviral agents (e.g., idoxuridine, adenine arabinoside,etc.); antimycotic agents (e.g., amphoteracin B, natamycin, pimaricin,flucytosine, nystantin, thimerosal, sulfamerazine, thiobendazole,tolnaftate, grisiofulvin, etc.); antiparasitic agents (e.g.,sulfonamides,pyrimethamine, clindamycin, etc.); and anti-inflammatoryagents (e.g., corticosteriods such as ACTH, hydrocortisone, prednisone,medrysone, beta methasone, dexamethasone, fluoromethalone,triamcinalone, etc.).

The following Examples are given for purposes of illustration and not byway of limitation on the scope of the invention.

6. EXAMPLE: PREPARATION OF SPLVS BY THE MONOPHASIC SOLVENT SYSTEMPROCESSES

In the subsections which follow, SPLVs were prepared by solubilizing aphospholipid in ethanol or other appropriate solvent, adding an aqueousphase and the material to be entrapped, sonicating the mixture at 54° C.while drying under nitrogen until a film formed. The film containingboth the lipid and the material to be entrapped was resuspendedin anaqueous buffer and agitated in order to form the SPLVs.

6.1. SPLVS Containing Tetracyclines

A sample containing 127 micromoles of egg phosphatidylcholine (EPC) inchloroform was taken to dryness in a round bottom flask. A 5 ml aliquotofethanol was added to the flask to resuspend the lipid. A solution (0.5ml) containing 100 mg of doxycycline monohydrate at approximately pH 7in physiologic saline was pipetted into the glass vessel containing theethanol solution of lipid. The monophase was placed in a bath sonicatortype 10536 (Laboratories Supplies Co., Inc.) for several minutes, (80kHz frequency; output 80 watts), at 54° C., while being dried to afilmby passing thereover a gentle stream of nitrogen.

To the film remaining 0.3-10.0 ml of physiologic saline was added andthe mixture was vortexed while being dried under nitrogen in order tosuspend the film and form the SPLVs. The preparation was centrifuged at10,000×g for 10 minutes to remove the non-entrapped doxycycline. Thiswash was repeated three times. The resulting pellet was suspended in 10ml of physiologic saline.

The same procedure was used to prepare SPLVs containing tetracycline bysubstituting tetracycline for doxycycline.

6.2. SPLVS Containing Gentamicin and Nafcillin

SPLVs containing both gentamicin and nafcillin were prepared asdescribed above with the following modifications: a 5 ml ethanolsolution containing100 mg EPC was prepared and the following twosolutions were added to the lipid-ethanol solution simultaneously: 100mg gentamicin sulfate in 0.15 ml PBS⁻ (phosphate buffered saline lackingdivalent cations) and 100 mg nafcillin in 0.15 ml PPS. The mixture wasevaporated at 54° C. and the SPLVs were formed as described above.

6.3. SPLVS Containing Gentamicin

SPLVs containing gentamicin (without nafcillin) were prepared by thesame procedure described in Section 6.2 except that 200 mg gentamicinsulfate in 0.3 ml PBS was added to the 5 ml ethanol-EPC solution.

6.4. SPLVS Containing Chloramphenicol

SPLVs containing chloramphenicol were prepared as described in Section6.1.except that chloramphenicol (crystalline) was substituted fordoxycycline.

6.5. SPLVS Containing Tobramycin

SPLVs containing tobramycin were prepared as follows: 300 mg EPC inchloroform rotoevaporated to dryness and resuspended in 30 ml ethanol.300mg tobramycin were suspended in PBS⁻ (PBS without divalent cations)toa final volume of 0.9 ml. The two solutions were mixed together byvortexing and then rotoevaporated to dryness in a water bath at 50°-55°C. The final film was resuspended in 10 ml PBS⁻, washed three times at10,000×g for 10 minutes centrifugation. Each supernatant was discardedand the pellet resuspended in 10 ml fresh PBS⁻. After the third wash,the final pellet was resuspended to a final volume of 10 ml, yielding afinal tobramycin concentration of 0.8 mg/ml as determined by standardmicrobiological assaytechniques.

6.6. SPLVS Containing Ticarcillin

SPLVs containing ticarcillin were prepared as follows: 2 grams EPC inchloroform was rotoevaporated to dryness and resuspended in 200 mlethanol. 2 grams ticarcillin sodium was added to the EPC-ethanol mixtureto form a solution. 6 ml PBS⁻ were then added to the solution andthesolution rotoevaporated to dryness in a water bath at 50°-55°C. Thefinal film was resuspended in 30 ml PBS⁻ and diluted to 100 ml in PBS⁻.The preparation was washed three times at 10,000×g for10 minutescentrifugation. Each supernatant was discarded and the pelletresuspended in fresh PBS⁻. After the final wash, the pellets were pooledand the final volume adjusted to 10 ml, yielding a final Ticarcillinconcentration of 10 mg/ml as determined by standard microbiologicalassay techniques.

6.7. SPLVS Containing Ticarcillin and Tobramycin

SPLVs containing both ticarcillin and tobramycin were prepared asfollows: 200 mg EPC in chloroform was rotoevaporated to dryness andresuspended in 20 ml ethanol. 200 mg ticarcillin sodium salt wasdissolved in this solution. 200 mg tobramycin was dissolved in 0.6 mlPBS⁻ and mixed with the above solution by vortexing. The mixture wasrotoevaporated to dryness in a water bath at 50°-55° C. The final filmwas resuspended in 10 ml PBS⁻ and the preparation washed three times at10,000×g for 10 minutes centrifugation. Each supernatant was discardedand the pellet resuspended to a final volume of 10 ml fresh PBS⁻. Afterthe final wash, the pellet was resuspended to a final volume of 10 ml inPBS⁻, yielding a final tobramycin concentration of0.8 mg/ml andticarcillin concentration of 0.8 mg/ml as determined by standardmicrobiological assay techniques.

6.8. Alternative Methods of Preparing SPLVS

SPLVs were prepared as follows: 127 micromoles of EPC in chloroform wastaken to dryness by rotoevaporation. The lipid was resuspended in 5 mlof ethanol and to this was added 0.2 ml water containing ³ H-inulin.Theresulting preparation was treated as follows to examine theencapsulation efficiency of the resulting liposomes:

(1) Vortexing the preparation while drying under nitrogen;

(2) Hand-shaking the preparation while drying under nitrogen;

(3) Drying under nitrogen with no concurrent agitation;

(4) Rotoevaporating under vacuum with no agitation;

(5) Sonicating while drying under nitrogen.

All techniques were carried out at a temperature range of between50°-60° C. To the dried preparations were added 10 ml of watercontaining ¹⁴ C-sucrose. All preparations were centrifuged at 10,000×gfor 10 minutes with three washes.

Final entrapment was determined by liquid scintillation countingtechniquesusing double channel counting. Values expressed as percententrapment meansthe percentage of radioactive material in the pelletedliposomes (cpm) relative to the initial amount of radioactive materialin the preparation (cpm). The results are shown in Table V.

                  TABLE V                                                         ______________________________________                                        EFFICIENCY OF ENTRAPMENT IN SPLVS                                             MADE BY ALTERNATIVE METHODS                                                                   Encapsulation Efficiency.sup.a                                Procedure         .sup.3 H-Inulin                                                                          .sup.14 C-Sucrose                                ______________________________________                                        (1) Vortexing while drying                                                                          31.0       2.3                                              under nitrogen                                                            (2) Hand-shaking while drying                                                                       29.7       2.4                                              under nitrogen                                                            (3) Stationary drying under                                                                         32.6       2.2                                              nitrogen                                                                  (4) Rotoevaporation   32.2       2.2                                          (5) Sonicating while drying                                                                         44.5       2.4                                              under nitrogen.sup.b                                                      ______________________________________                                         .sup.a Figures presented represent percent entrapment of the starting         volumes used.                                                                 .sup.b Preferred embodiment.                                             

6.9. Preparation of SPLVS Using Various Solvent Systems

The following example shows the encapsulation efficiency of SPLVs thatare prepared in different solvent systems. The criteria used for theevaluation of the solvents tested in this example were the following:(1) 5 ml of the organic solvent must form a monophasic solution with 0.2ml aqueous solvent and (2) EPC must be soluble in the monophase. Ofcourse ifless lipid is used to make the SPLVs the volumes used in thetest would be adjusted accordingly.

Seven organic solvents were evaluated according to the above criteriaand the results are shown in Table VI.

                  TABLE VI                                                        ______________________________________                                        SELECTION OF SOLVENTS                                                                      Criterion 1   Criterion 2                                                     5 ml of solvent                                                                             At 50°-60° C.,                                    are miscible with                                                                           solvent, lipid and                                 Solvent      0.2 ml H.sub.2 O                                                                            H.sub.2 O are miscible                             ______________________________________                                        Ethanol      Yes           Yes                                                Acetone      Yes           Yes                                                Dimethylformamide                                                                          Yes           No                                                 DMSO         Yes           No                                                 Acetonitrile No            Not Done                                           2-Propanol   Yes           Yes                                                Methanol     Yes           Yes                                                ______________________________________                                    

These results indicate that four of the solvents examined are suitableto use as solvent for preparation of SPLVs (i.e., ethanol, acetone,2-propanol or methanol).

In order to determine the encapsulation efficiencies achieved when usingthe four solvent systems, a sample of 127 micromoles of EPC inchloroform was rotoevaporated to dryness in a round bottom flask, thenresuspended inone of the following organic solvents: ethanol, acetone,2-propanol, or methanol. to this preparation was added 0.2 ml of anaqueous phase containing ³ H-inulin. This monophase was sonicated at50°-60° C., and dried under nitrogen. The resulting film wasresuspendedin 10 ml of water containing ¹⁴ C-sucrose after being subjected tocentrifugation three times at 10,000×g. Final entrapment of ³ H-inulinand ¹⁴ C-sucrose were determined by dual channel liquid scintillationtechnique (Dual Beckman LS 6800). The results are shown in Table VII.

                  TABLE VII                                                       ______________________________________                                        ENCAPSULATION EFFICIENCIES OF VARIOUS                                         SOLVENT SYSTEMS                                                                            Encapsulation Efficiency.sup.a                                   Organic Solvent                                                                              .sup.3 H-Inulin.sup.b                                                                   .sup.14 C-Sucrose.sup.c                              ______________________________________                                        Ethanol        45.8      2.8                                                  Acetone        38.3      2.3                                                  2-Propanol     23.7      1.3                                                  Methanol       44.5      2.4                                                  ______________________________________                                         .sup.a Values are expressed as percent entrapped meaning the proportion o    radioactive material in the pelleted liposomes (cpm) relative to the           starting amount of radioactive material (cpm) added to the preparation.       .sup.b Added to the monophase.                                                .sup.c Added to the aqueous resuspension buffer.                         

7. EXAMPLE: PREPARATION OF SPLVS BY THE EMULSIFICATION PROCESSES

As explained in Section 5.1.2. one basic process for preparing SPLVsinvolves dissolving a lipid or mixture of lipids into an organicsolvent, adding an aqueous phase and the material to be encapsulated,and sonicating the mixture while sonicating by any evaporativetechnique. The SPLVs used in all of the examples contained herein wereprepared as described in the following subsections (however any processwhich yields SPLVs may be used).

5.7.1. SPLVS Containing Antibiotics

A 5 ml diethyl ether solution of 100 mg lecithin was prepared. Themixture was placed in a round-bottom flask. Then a solution (0.3 ml)containing 100 mg of streptomycin sulfate at pH 7.4 in 5 mM HEPES(4-[2-Hydroxyethyl]piperazino 2-ethane sulfonic acid)/0.0725MNaCl/0.0725M KCl was pipetted into the glass vessel containing thediethyl ether solution of lipid. The mixture was placed in a bathsonicator (Laboratory Supplies Co., Inc.) type 10536 for severalminutes, (80 kHz frequency:output 80 watts) while being dried to aviscous paste by passing thereover a gentle stream of nitrogen.

To the viscous paste remaining was added 10 ml of 5 mM HEPES. TheresultingSPLV preparation, containing streptomycin, was suspended in thebuffer solution, shaken for several minutes on a vortex mixer, and freedof non-encapsulated streptomycin by centrifuging at 12,000×g for 10minutes at 20° C. The resulting cake was suspended in 0.5 ml of 5 mMHEPES.

The procedure described above was followed except that streptomycin wassubstituted by each one of the following: dihydrostreptomycin,gentamicin sulfate, ampicillin, tetracyline hydrochloride, clindamycinand kanamycin.

7.2. Preparation of SPLVs Containing Gentamicin or Nafcillin

A 5 ml diethyl ether solution of 100 mg egg phosphatidylcholine (EPC, oregg lecithin) was prepared. The mixture was placed in a round bottomflask. Then a solution (0.3 ml) containing 200 mg of gentamicin ornafcillin in phosphate buffered saline (PBS, pH 7.2) was pipetted intotheflask containing the diethyl ether solution of lipid. The mixture wasplaced in a bath sonicator (Laboratory Supplies Co., Inc., type 10536)forseveral minutes (80 kHz frequency; output 80 watts) while being driedto a viscous paste by passing a gentle stream of nitrogen over themixture.

To the viscous part remaining, 10 ml of PBS was added. The resultingSPLV preparation containing either nafcillin (SPLV/Naf) or gentamicin(SPLV/Gent) was suspended in PBS, shaken and freed of nonencapsulatedantibiotic by centrifugation at 12,000×g for 10 minutes at 20° C. Theresulting pellet was washed one more time and resuspended in 0.5 ml PBS.

7.3. Preparation of SPLVs Containing Both Gentamicin and Nafcillin

In order to prepare SPLVs containing both nafcillin and gentamicin, theprocedure described above was followed with the following modifications:after the EPC was dispersed in diethyl ether, two solutions, one of eachantibiotic, were added quickly and simultaneously, each solutionconsistedof 100 mg antibiotic (nafcillin or gentamicin) dissolved in0.15 ml PBS. After the addition of the two solutions, the preparationwas sonicated, evaporated, and washed two times as previously described.The resulting SPLVs entrapped both gentamicin and nafcillin(SPLV/Gent-Naf).

7.4. SPLVS Containing Gentamicin and Clindamycin

SPLVs containing both gentamicin and clindamycin were prepared asdescribedin Section 7.3 except 100 mg clindamycin was used in place ofnafcillin.

7.5. SPLVS Containing Other Membrane Constituents

The process described in Section 7.1 was followed except that any one ofthe following was added with the egg lecithin: (1) phosphatidic acid togive a molar ratio of 8:2 (lecithin:dicetylphosphate); (2) stearylaminetogive a molar ratio of 8:2 (lecithin: stearylamine); cholesterol andstearylamine to give a molar ratio of 7:2:1(lecithin:cholesterol:stearylamine); and (3) phosphatidic acid andcholesterol to give a molar ratio of 7:2:1 (lecithin:phosphatidicacid:cholesterol).

7.6. SPLVS Containing Pilocarpine

The procedure of Section 7.1. was followed except that the antibioticstreptomycin was replaced with pilocarpine.

7.7. SPLVS Prepared With and Without BHT

Undistilled ether contains an anti-oxidant, 1 μg/ml butylhydroxytoluene(BHT), for storage purposes. The procedure described in Section 7.1. wasfollowing using undistilled ether as the solvent in order to incorporateBHT into the SPLV preparation.

In order to prepare SPLVs without incorporation of BHT, the proceduredescribed in Section 7.1. was followed using distilled ether as thesolvent.

8. EXAMPLE: SPLV MEDIATED DELIVERY IN VITRO

In the following example, SPLV mediated delivery of antibiotics tomacrophages in culture was demonstrated.

Peritoneal macrophages were obtained by peritoneal lavage from C57BLKadultmale mice and suspended in minimal essential medium (M.E.M.) pH 7.2containing 10% heat-inactivated fetal calf serum. Cells were suspendedat a concentration of 1×10⁶ cells per ml in 96-well tissueculturedishes. To cultures containing adherent peritoneal macrophages,were added B. canis at concentrations of 1×10⁶ CFU (colony formingunits) per ml. After 12 hours, bacteria not engulfed by peritonealmacrophages were removed by repeated washings with M.E.M. After washingof peritoneal macrophage cultures, they were divided into 5 groups, eachcontaining 12 replicate cultures per group. Group 1, designatedControls, received no treatment. Group 2 received aqueous streptomycinsulfate at a concentration of 1 mg/ml. Group 3 received buffer-filledSPLVs. Group 4 received aqueous streptomycin sulfate (1 mg/ml) pluspreformed buffer-filled SPLVs. Group 5 received SPLVs containingstreptomycin sulfate (1 mg/ml). After 24 hours, supernatants wereremoved by repeated washings and peritoneal macrophages were disruptedby repeated freezing and thawing. Serial dilutions of disruptedmacrophages were plated onto brucella agar and, after 4 days, survivingB. canis were determined by limiting dilution techniques. Results shownin Table VII indicate that SPLV-entrapped streptomycin was totallyeffective in killing and eliminating the intracellular B. canisinfection in vitro.

The experiment was repeated with B. abortus exactly as described aboveexcept that peritoneal macrophages were obtained by peritoneal lavagefromadult female albino guinea pigs. Results are also shown in TableVIII.

                  TABLE VIII                                                      ______________________________________                                        COLONY-FORMING UNITS OF INTRACELLULAR                                         BRUCELLA ISOLATED AFTER TREATMENT OF                                          INFECTED MACROPHAGES WITH SPLVS CON-                                          TAINING STREPTOMYCIN                                                                      B. canis.sup.a                                                                            B. abortus.sup.b                                      ______________________________________                                        Controls       2.6 ± 1.13 × 10.sup.3                                                            3.1 ± 0.81 × 10.sup.4                     Buffer-filled 2.82 ± 0.10 × 10.sup.3                                                            2.9 ± 0.17 × 10.sup.4                     SPLVs                                                                         Free          3.11 ± 0.40 × 10.sup.3                                                            3.3 ± 0.25 × 10.sup.4                     Streptomycin.sup.c                                                            Streptomycin  2.76 ± 0.20 × 10.sup.3                                                            2.8 ± 0.42 × 10.sup.4                     Plus Buffer-                                                                  filled SPLVs.sup.c                                                            SPLV-Entrapped                                                                              0            0                                                  Streptomycin.sup.c                                                            ______________________________________                                         .sup.a Colony forming units (CFU) of B. canis (mean ± SD of 12             replicates) isolated from equal numbers of previously infected mouse          (C57B1k) peritoneal macrophages.                                              .sup.b CFU of B. abortus (mean ± SD of 12 replicates) isolated from        equal numbers of previously infected guinea pig peritoneal macrophages.       .sup.c Concentration of streptomycin 1 mg/ml.                            

9. EXAMPLE: TREATMENT OF INTRACELLULAR INFECTIONS

The following examples demonstrate how SPLVs can be used in treatingintracellular infections. The data presented demonstrates: (1) theeffectiveness of using antibiotics encapsulated in SPLVs in thetreatment of disease and (2) the greater efficiency which is obtained byadministering multiple doses of the SPLV preparation. A comparison ofMLVsto SPLVs as vehicles used in the protocols is described.

Brucellosis causes worldwide economic and public health problems.Brucellosis is caused by Brucella spp. It is adapted to many mammalianspecies, including man, domestic animals and a variety of wild animals.Six Brucella spp. cause brucellosis in animals; they are B. abortus, B.canis, B. melitensis, B. neotomae, B. ovis and B. suis. Both domesticand wild animals serve as reservoirs for potential spread of brucellosisto other animals and man.

Such infections cannot be cleared with antibiotics because theinfectious organisms reside within the cells of the reticuloendothelialsystem and are highly resistant to bactericidal activities ofantibiotics. The quantity of antibiotics required and the length oftreatment results in either toxic effects on the animal or anunacceptably high concentration of the antibiotic in the tissues of theanimal. The further difficulty in treating this disease is that thetreatment has to be completely effectivesince any remaining infectionwill simply spread and the cycle commences once again. The economicimpact of such diseases is demonstrated by the millions of dollars ofvaluable cattle which are lost each year due to spontaneous abortion.The only potential way to combat such infectious outbreaks is toquarantine and then slaughter the animals.

The examples which follow comprise incorporating an antibiotic intoSPLVs, and then administrating the encapsulated active substance to theanimals by inoculating the infected animals intraperitoneally.

9.1. Effect of a Single Treatment of B. Canis Infection UsingSPLV-entrapped Antibiotic

In the following experiment, SPLVs were prepared by the emulsificationprocess as described in Section 7. Eighty adult male Swiss mice wereinfected intraperitoneally (I.P.) with B. canis ATCC 23365 (1×10⁷ CFU)and divided into 8 groups of 10 mice each. Seven days post-inoculationwith B. canis, groups were treated as follows: Group1, designatedControls, received no treatment; Group 2 received buffer-filled SPLVs(0.2 ml I.P.); Group 3 received aqueous streptomycin sulfate (1 mg/kgbody weight in a total administration of 0.2 ml I.P.); Group 4 receivedaqueous streptomycin sulfate (5 mg/kg body weight) in a totaladministration of 0.2 ml I.P.; Group 5 received aqueousstreptomycinsulfate (10 mg/kg body weight) in a total administration of0.2 ml I.P.; Group 6 received SPLVs containing streptomycin sulfate (1mg/kg body weight) in a total administration of 0.2 ml I.P.; Group 7received SPLVs containing streptomycin sulfate (5 mg/kg body weight) ina total administration of 0.2 ml I.P.; and Group 8 received SPLVscontaining streptomycin sulfate (10 mg/kg body weight) in a totaladministration of 0.2 ml I.P.. On day 14 post-inoculation with B. canis,all animals were sacrificed and spleens were removed aseptically.Spleens were homogenized and serially diluted onto brucella agar todetermine the number of surviving B. canis in spleens after treatment.Results after 4 days incubation are shown in Table IX.

                  TABLE IX                                                        ______________________________________                                        EFFECT OF A SINGLE TREATMENT.sup.a OF B. CANIS                                INFECTED MICE WITH VARIOUS CONCENTRATIONS                                     OF FREE OR SPLV-ENTRAPPED STREPTOMYCIN                                        ______________________________________                                               Colony-Forming Units B. Canis Per Spleen.sup.b                                  No Treatment    Buffer-Filled SPLVs                                  ______________________________________                                        Control  3.46 × 10.sup.6 ± 2.7 × 10.sup.6                                               4.1 × 10.sup.6 ± 0.66                       ______________________________________                                                                 × 10.sup.6                                     Strepto-                                                                      mycin Con-                                                                    centration                                                                    (mg/kg body                                                                            Free            SPLV-Entrapped                                       weight)  Streptomycin    Streptomycin                                         ______________________________________                                        1         1.5 ± 0.45 × 10.sup.6                                                               1.01 ± 0.25 × 10.sup.3                      5        2.12 ± 1.71 × 10.sup.5                                                                1.52 ± 0.131 × 10.sup.4                    10       9.66 ± 3.68 × 10.sup.4                                                               1.32 ± 1.00 × 10.sup.4                      ______________________________________                                         .sup.a I.P. injection in total of 0.2 ml (sterile saline).                    .sup.b Surviving B. canis was determined as the number of CFU isolated pe    spleen and is expressed as mean ± S.D. of 10 animals per experiment         (triplicate experiments).                                                

9.2. Effect of Multiple Treatment of B. Canis Infection UsingSPLV-entrapped Antibiotic

In the following experiments, SPLVs were prepared by the emulsificationprocess described in Section 7.

Eighty adult male Swiss mice were infected with B. canis ATCC 23365(1×10⁷ CFU, I.P.) and divided into 8 groups of 10 mice each. Seven daysand 10 days post-inoculation with B. canis, groups were treatedasfollows: Group 1, designated controls, received no treatment; Group 2received buffer-filled SPLVs (0.2 ml, I.P.); Group 3 received aqueousstreptomycin sulfate (1 mg/kg body weight) in a total administration of0.2 ml, I.P.); Group 4 received aqueous streptomycin sulfate (5 mg/kgbodyweight) in a total administration of 0.2 ml, I.P.; Group 5 receivedaqueousstreptomycin sulfate (10 mg/kg body weight) in a totaladministration of 0.2 ml, I.P.; Group 6 received SPLVs containingstreptomycin sulfate (1 mg/kg body weight) in a total administration of0.2 ml, I.P.; Group 7 received SPLVs containing streptomycin sulfate (5mg/kg body weight) in a total administration of 0.2 ml, I.P.; and Group8 received SPLVs containing streptomycin sulfate (10 mg/kg body weight)in a total administration of 0.2 ml, I.P. On day 14 post-inoculationwith B. canis, all animals were sacrificed and spleens were removedaseptically. Spleens were homogenized and serially diluted onto brucellaagar to determine the number of surviving B. canis in spleens aftertreatment. Results after 4 days incubation are shown in FIG. 18.

The results of various two-stage treatment regimens on B. canisinfections in vivo presented in FIG. 18, demonstrate that in groupsreceiving aqueousstreptomycin 7 and 10 days post-inoculation, verylittle reduction in surviving B. canis in spleens was observed. Only ingroups receiving SPLV-entrapped streptomycin at a concentration of 10mg/kg body weight administered on day 7 and 10 post-inoculation were allviable bacterial eliminated from spleens of infected animals.

In addition to the experiment described above, various tissues from B.canis infected mice after two treatments with SPLV-entrappedstreptomycin were sampled as follows:

Thirty adult male Swiss mice were inoculated with B. canis ATCC 23365(1×10⁷ CFU, I.P.). Seven days post-inoculation animals were divided into3 groups of 10 mice each. Group 1, designated controls, received notreatment; Group 2 received (on days 7 and 10 post-inoculation) aqueousstreptomycin sulfate (10 mg/kg body weight) in each administration of0.2 ml), I.P.; Group 3 received (on days 7 and 10 post-inoculation)SPLVs containing streptomycin sulfate (10 mg/kg body weight) in eachadministration of 0.2 ml, I.P. On days 14 to 75 post-inoculation with B.canis, all animals were sacrificed and the following organs removedaseptically, homogenized and serially diluted onto brucella agar forisolation of B. canis: heart, lungs, spleen, liver,kidneys, testes.After 4 days incubation, results of surviving B. canis perorgan areshown in FIG. 19.

Results of samplings of various tissues in B. canis infected mice aftertwotreatment regimens with streptomycin presented in FIG. 19,demonstrated that in animals treated with SPLV-entrapped streptomycin,all tissues sampled from 14 to 75 days post-inoculation with B. caniswere totally free of any viable B. canis organisms. In animals untreatedor treated with aqueous streptomycin in concentrations andadministration schedules identical to those receiving SPLV-entrappedstreptomycin, viable B. canis organisms could be isolated in all tissuessampled from 14 to 75 days post-inoculation with B. canis.

In the following experiment, SPLVs were prepared by the monophasicsolvent system method as described in Section 6.

Twenty adult female Swiss Webster mice were infected with B. canis ATCC23365 (5×10⁶ colony forming units, CFU) intraperitoneally (I.P.) anddivided into 2 groups of 10 mice each. Seven days and 10 dayspost-inoculation with B. canis, groups were treated as follows: Group 1,designated controls, received no treatment, Group 2 received SPLVscontaining gentamicin (10 mg/kg body weight) in a total volume of 0.3ml, I.P. On day 17 post-inoculation with B. canis, all animals weresacrificedand spleens removed aseptically. Spleens were homogenized andserially diluted onto brucella agar to determine the number of survivingB. canis in spleens after treatment. Results after 3 days incubation areshown in Table X.

The results of the two-stage treatment regimens on B. canis infectionsin vivo presented in Table X, demonstrate that in groups receivingSPLV-entrapped gentamicin at a concentration of 10 mg/kg of body weightadministered on days 7 and 10 post-inoculation all viable bacteria wereeliminated from spleens of infected animals.

                  TABLE X                                                         ______________________________________                                        EFFECTIVENESS OF SPLVS CONTAINING                                             GENTAMICIN ON KILLING OF B. CANIS                                             IN VIVO AFTER TWO TREATMENTS.sup.a                                                       Colony-Forming                                                                Units per Spleen.sup.b                                             ______________________________________                                        Control      2.20 ± 0.26 × 10.sup.4                                  SPLVs.sup.c  0                                                                ______________________________________                                         .sup.a Intraperitoneal injections, 10 mg/kg body weight, were spaced at 3     day intervals. Controls received no treatment.                                .sup.b Surviving B. canis was determined as the number of CFU isolated pe    spleen and is expressed as the mean ± S.D. of 20 cultures.                  .sup.c Egg phosphatidylcholine to gentamicin ratios were 100 mg lipid to      30 mg gentamicin.                                                        

9.3. Effectiveness of Treatments Using MLVS as Compared to SPLVS

In the following experiment, SPLVs were prepared by the emulsificationprocess as described in Section 7. Fifteen adult male Swiss mice wereinoculated with B. canis ATCC 23365 (1×10⁷ CFU, I.P.). Seven dayspost-inoculation animals were divided into 3 groups of 5 mice each.Group 1, designated Controls, received no treatment; Group 2 received(on days 7 and 10 post-inoculation) MLVs containing streptomycin sulfate(10 mg/kg body weight, I.P.). MLVs were prepared by conventionaltechniques using 100 mg egg phosphatidylcholine (EPC) and 2 ml ofsterile HEPES containing streptomycin sulfate (100 mg/ml). The lipid tostreptomycin sulfate ratio was 100 mg EPC to 28 mg streptomycin sulfatein the 2 ml final MLV suspension; Group 3 received (on days 7 and 10post-inoculation)SPLVs containing streptomycin sulfate (10 mg/kg bodyweight, I.P.) preparedas described in Section 7.1. with the followingmodifications: 100 mg EPC were used, and 0.3 ml of HEPES containing 100mg streptomycin sulfate. The lipid to streptomycin sulfate ratio inSPLVs was 100 mg EPC to 28 mg streptomycin sulfate in a 2 ml finalsuspension. On day 14 post-inoculation with B. canis, all animals weresacrificed and spleens were removed aseptically, homogenized andserially diluted onto brucella agar for isolation of B. canis. Resultsof surviving B. canis per organ after 4 days incubation are shown inTable XI.

                  TABLE XI                                                        ______________________________________                                        COMPARISON OF MLVS AND SPLVS CONTAINING                                       STREPTOMYCIN SULFATE ON KILLING OF B. CANIS                                   IN VIVO AFTER TWO TREATMENTS.sup.a                                                      Colony-Forming Units                                                          B. Canis per Spleen.sup.b                                           ______________________________________                                        Control     2.7 ± 1.0 × 10.sup.4                                     MLVs.sup.c  1.8 ± 0.4 × 10.sup.4                                     SPLVs.sup.c 0                                                                 ______________________________________                                         .sup.a Intraperitoneal injections, 10 mg/kg body weight, were spaced at 3     day intervals. Controls received no treatment.                                .sup.b Surviving B. canis was determined as the number of CFU isolated pe    spleen and is expressed as the mean ± S.D. of 5 animals per group           (duplicate determinations per animal).                                        .sup.c Egg phosphatidylcholine to streptomycin sulfate ratios were 100 mg     lipid to 28 mg streptomycin sulfate.                                     

9.4. Effect of Various SPLV-entrapped Antibiotics on Treatment ofInfection

In the following experiment, SPLVs were prepared by the emulsificationmethod as described in Section 7.

Fifty adult male Swiss mice were inoculated with B. canis ATCC 23365(1×10⁷ CFU, I.P.). Seven days post-inoculation, animals were dividedinto 10 groups of 5 mice each. Group 1, designated controls, received notreatment; Group 2 received buffer-filled SPLVs (0.2 ml, I.P.)on days 7and 10 post-inoculation; Groups 3, 4, 5 and 6 received aqueousinjections (0.2 ml I.P.) of dihydrostreptomycin, gentamicin, kanamycinor streptomycin 10 mg/kg body weight, I.P. on days 7 and 10post-inoculation (N.B. Each of these antibiotics have been shown to killB. canis in vitro). Groups 7, 8, 9, and 10 received SPLVs containingdihydrostreptomycin, gentamicin, kanamycin, or streptomycin at 10 mg/kgbody weight on days 7 and 10 postinoculation. On day 14 post-inoculationwith B. canis, all animals were sacrificed and spleens were removedaseptically, homogenized and serially diluted onto brucella agar for atisolation of B. canis. Results of surviving B. canis per organ after 4days incubation are as shown in Table XII.

The results from tests of various antibiotics on B. canis infected micepresented in Table XII demonstrate that antibiotics which are effectiveinkilling B. canis in vitro (i.e., in suspension culture) are also onlyeffective in killing B. canis infections in vivo when they areencapsulated within SPLVs. Animals receiving either aqueous antibiotics,buffer-filled SPLVs or no treatment were in no case cleared of survivingB. canis in isolated spleen tissues.

9.5. Treatment of Dogs Infected with B. Canis

In the following experiment, SPLVs were prepared by the emulsificationprocess as described in Section 7. Adult female beagles were inoculatedwith B. canis ATCC 23365 (1×10⁷ CFU) orally and vaginally. Seven dayspost-inoculation dogs were divided into 3 groups. Group 1, designatedcontrol, received no treatment; Group 2 received (on days 7 and10post-inoculation) aqueous streptomycin sulfate at 10 mg/kg body weight(each administration was 5.0 ml, I.P.). Group 3 received (on days 7 and10post-inoculation) SPLVs containing streptomycin sulfate at 10 mg/kgbody weight (each administration was 3.0 ml, I.P.).

                  TABLE XII                                                       ______________________________________                                        COMPARISON OF VARIOUS ANTIBIOTICS ON                                          KILLING OF B. CANIS IN VIVO AFTER                                             TWO TREATMENTS.sup.a                                                                     Colony-Forming Units B. Canis                                                 Per Spleen.sup.b                                                              Aqueous     SPLV-Entrapped                                                    Solutions   Antibiotic                                             ______________________________________                                        Untreated    3.93 ± 1.51 × 10.sup.6                                                             4.66 ± 0.87 × 10.sup.6                    Antibiotic.sup.c                                                              Dihydrostreptomycin                                                                        1.13 ± 0.30 × 10.sup.5                                                             0                                                  Gentamicin   7.06 ± 2.53 × 10.sup.5                                                             0                                                  Kanamycin    2.72 ± 0.91 × 10.sup.5                                                             0                                                  Streptomycin 1.01 ± 0.17 × 10.sup.5                                                             0                                                  ______________________________________                                         .sup.a Intraperitoneal treatments, 10 mg/kg body weight, were spaced at 3     day intervals. Controls received no treatment.                                .sup.b Surviving B. canis per organ was determined as the number of CFU       isolated per spleen and expressed as the mean ± S.D. of 5 animals per      groups (duplicate determinations per animal).                                 .sup.c Antibiotics effective in killing B. canis in suspension culture.  

                                      TABLE XIII                                  __________________________________________________________________________    RESULTS OF CULTURES AND SEROLOGICAL TESTING IN B. CANIS                       INFECTED DOGS SUBJECTED TO A TWO TREATMENT ANTI-                              BIOTIC REGIMEN.sup.a                                                          Days After                   SPLV-                                            Infection                    Entrapped.sup.c                                  with    Control   Streptomycin.sup.b                                                                       Streptomycin                                     B. Canis                                                                              R  M  B V R  M  B V  R  M  B V                                        __________________________________________________________________________    Pre-treatment                                                                 0       0  0  0 0 0  0  0 0  0  0  0 0                                        2       ND ND + + ND ND + 0  ND ND + +                                        4       ND ND + + ND ND + +  ND ND + +                                        Post-treatment                                                                8       0  0  0 + 0  0  + 0  0  0  0 0                                        10      0  0  0 + 0  0  0 +  0  0  0 0                                        21      1.5                                                                              2  + + 1  2  + +  0  0  0 0                                        __________________________________________________________________________     .sup.a R (rapid slide agglutination test) indicates the reciprocal of         serum titer to B. canis antigen (×10.sup.2); 0 = no detectable          titer.                                                                       M (2mercaptoethanol tube agglutination test) indicates the reciprocal of       serum titer to B. canis antigen (×10.sup.2); 0 = no detectable          titer.                                                                       In B (blood culture) and V (vaginal culture) on brucella agar: + =             detection of greater than or equal to 1 CFU; 0 = no colonies detected.        Controls received no treatment.                                               .sup.b Streptomycin sulfate (aqueous) 10 mg/kg body weight, I.P.              .sup.c SPLVs containing streptomycin sulfate 10 mg/kg body weight, I.P.  

Vaginal swabbings of dogs and heparinized blood samples were collectedat regular intervals before, during, and at the termination of thestudy. These were cultured on brucella agar in order to isolate B.canis. Resultsare shown in Table XIII. Serum samples were collectedbefore, during, and at the termination of the study for determinationsof serum antibody against B. canis. These results are also shown inTable XIII.

Twenty-one days post-inoculation with B. canis, all animals wereeuthanized. The following tissues were removed aseptically, homogenizedand serially diluted onto brucella agar for isolation of B. canis:heparinized blood, vaginal exudate, lungs, spleen, synovial fluid,uterus,ovary, popliteal lymph nodes, salivary glands, tonsils,mediastinal lymph nodes, mesenteric lymph nodes, bone marrow,superficial cervical lymph nodes, and auxiliary lymph nodes. Results ofsurving B. canis per tissue after 4 days incubation are shown in TableXIV.

Results of culture and serologic tests of dogs infected with B. canisbefore, during, and after two-stage antibiotic administration arepresented in Table XVI. All animals were serologically negative forprevious exposure to B. canis as measured by negative serum titers, andwere culture negative from blood cultures and cultures of vaginalswabbings. All animals were noted to be culture positive for both bloodand vaginal cultures prior to treatments on days 7 and 10. Dogs treatedwith aqueous streptomycin or dogs receiving no treatment remainedculture positive for blood and vaginal cultures during post-treatmentperiods prior to termination on day 21. Group 3, which receivedliposomes containing streptomycin, became culture negative one day afterthe first treatment and remained negative throughout post-treatmentperiod.

                  TABLE XIV                                                       ______________________________________                                        RESULTS OF CULTURES FROM TISSUE SAMPLES                                       IN B. CANIS INFECTED DOGS SUBJECTED TO A                                      TWO TREATMENT ANTIBIOTIC REGIMEN.sup.a                                                    SPLVs                                                                         Containing                                                        Tissue.sup.b                                                                              Streptomycin.sup.c                                                                       Streptomycin.sup.d                                                                        Control.sup.e                              ______________________________________                                        Whole blood 0          +           +                                          Vaginal swab                                                                              0          +           +                                          Lungs       0          +           +                                          Spleen      0          +           +                                          Synovial fluid                                                                            N.D.       0           0                                          Uterus      0          +           +                                          Ovary       0          +           +                                          Popliteal lymph nod                                                                       N.D.       +           +                                          Salivary gland                                                                            0          0           0                                          Tonsil      0          +           +                                          Mediastinal lymph                                                                         0          N.D.        +                                          node                                                                          Mesenteric lymph                                                                          N.D.       0           0                                          node                                                                          Bone marrow 0          +           +                                          Superficial N.D.       N.D.        +                                          cervical lymph node                                                           Axillary lymph                                                                            0          +           +                                          node                                                                          ______________________________________                                         .sup.a Animals treated on day 7 and 10 postinfection.                         .sup.b Samples taken at necropsy were serially diluted on brucella agar;      = equal to or greater than 1 CFU; 0 = no colonies.                            .sup.c SPLVs containing streptomycin sulfate, 10 mg/kg body weight, I.P.      .sup.d Streptomycin sulfate (aqueous), 10 mg/kg body weight, I.P.             .sup.e Controls received no treatment.                                   

Dogs which received no treatment or aqueous streptomycin developeddetectable serum titers against B. canis antigens by day 21post-inoculation, while those treated with SPLVs containing antibioticsondays 7 and 10 post-inoculation did not develop any detectable antibodyto B. canis antigen.

Results from isolation of B. canis from infected dogs treated withtwo-stage antibiotic administration which are presented in Table XVIIdemonstrate that in dogs, only treatment with SPLVs containingstreptomycin was effective in eliminating any viable B. canis in alltissues from all organ samples.

9.6. Treatment of B. Abortus in Guinea Pigs

In the following experiment, SPLVs were prepared by the emulsificationmethod as described in Section 7.

Fifteen adult female guinea pigs were inoculated with B. abortus ATCC23451(1×10⁷ CFU, I.P.). Seven days post-inoculation animals were dividedinto 3 groups of 5 animals each. Group 1, designated Controls, receivedno treatment. Group 2 received aqueous streptomycin sulfate,I.P.injections (0.2 ml) at 10 mg/kg body weight on day 7 and 10post-inoculation with B. abortus. Group 3 received SPLVs containingstreptomycin sulfate I.P. injections (0.2 ml) at 10 mg/kg body weight ondays 7 and 10 post-inoculation with B. abortus. On day 14post-inoculationwith B. abortus, all animals were sacrificed and spleenswere removed, aseptically homogenized and serially diluted onto brucellaagar for isolation of B. abortus. Results of surviving B. abortus perspleen after 4 days incubation, are shown in FIG. 20. Only SPLVscontaining streptomycin were effective in eliminating B. abortusresiding within guinea pig spleen. In animals receiving aqueousstreptomycin or no treatment, viable B. abortus bacteria were beidentified.

9.7. Treatment of B. Abortus Infection in Cows

In the following experiment, SPLVs were prepared by the emulsificationprocess as described in Section 7.

Nine heavily infected animals were utilized in this experiment. B.abortus bacterial isolations from milk and vaginal swabbings became andremained negative for six weeks following treatment with SPLVscontaining streptomycin. When infection reoccurred in these animals,bacterial isolations were found only in quadrants of the udder whichwere positive prior to treatment.

Nine cross-bred (Hereford-Jersey-Brangus), 22-month old, non-gravid,confirmed B. abortus culture-positive cows were used. At least 4 monthsprior to the initiation of the study, the animals were experimentallychallenged per conjunctivum with 1×10⁷ CFU of B. abortus Strain2308during mid-gestation, which resulted in abortion and/or B. abortusculture positive lacteal or uterine secretions and/or fetal tissues.

Cows were maintained in individual isolation stalls and separated intothree groups. Treatment comprised a two-dose regimen, spaced 3 daysapart,as follows: (1) 3 cows were injected intraperitoneally withphysiological saline. (2) 3 cows were injected intraperitoneally withaqueous antibiotic(streptomycin at 10 mg/kg body weight) plus preformedbuffer-filled SPLVs. (3) 3 cows were injected intraperitoneally withSPLV-entrapped streptomycin (10 mg/kg body weight). The total volume perinjection was 100 ml per animal.

During the first 2 months duplicate bacteriologic cultures of lactealand uterine secretions were performed weekly providing secretions wereobtainable. Then, all cows were euthanatized with an overdose of sodiumpentabarbitol, and the following organs were collected in duplicate forbacteriologic cultures: (1) lymph nodes: left and right atlantal, leftandright suprapharyngeal, left and right mandibular, left and rightparotid, left and right prescapular, left and right prefemoral, left andright axillary, left and right popliteal, left and right internal iliac,left and right supramammary, left and right renal, bronchial,mediastinal, mesenteric, and hepatic; (2) glands: all four quarters ofmammary gland, left and right adrenal glands and thymus (if present);(3) organs and other tissues: spleen, liver, left and right horn ofuterus, cervix, vagina, kidney and tonsil.

After necropsy, all tissues were frozen and maintained at -70° C. whilein transport. Tissues were thawed, alcohol flamed, and asepticallytrimmed prior to weighing. Once weights were recorded (0.2 to 1.0grams), the tissue was homogenized in 1 ml of sterile saline andserially diluted with sterile saline to 1:10-10 of initial homogenatesuspension. Aliquots (20 μl) of each dilution from serial suspensionswere plated onto brucella agar and placed in 37° C. incubation.Duplicate determinations were performed for each tissue.

Plates were read daily and scored for bacterial growth. All coloniesappearing prior to 3 days were isolated, passaged, and gram stained todetermine identity. On days 5, 6 and 7 during incubation colonies withmorphology, growth, and gram staining characteristics consistent with B.abortus were counted; the CFU per gram tissue was then determined.Representative colonies were repassaged for bacterial confirmation of B.abortus.

                  TABLE XV                                                        ______________________________________                                        RESULTS OF CULTURES FROM TISSUE                                               SAMPLES OF B. ABORTUS INFECTED COWS                                                                SPLV-Entrapped                                                       Untreated                                                                              Streptomycin                                             Tissue        Control    1       2     3                                      ______________________________________                                        Adrenal gland L                                                                             0          0       0     0                                      Adrenal gland R                                                                             ++         0       0     +                                      Atlantal LN R ++         +       0     +                                      Atlantal LN L 0          0       0     +                                      Axillary LN R +++        0       +     0                                      Axillary LN L ++         0       0     0                                      Bronchial LN  0          0       0     0                                      Cervix        0          0       0     0                                      Hepatic LN    ++++       0       0     0                                      Horn of Uterus L                                                                            0          0       0     +                                      Horn of Uterus R                                                                            0          0       0     0                                      Int. Illiac LN R                                                                            ++         0       0     0                                      Int. Illiac LN L                                                                            ++++       0       +     0                                      Kidney        0          0       0     0                                      Liver         0          0       0     0                                      Lung          0          0       0     0                                      Mammary Gland LF                                                                            0          +       +     0                                      Mammary Gland LR                                                                            0          0       0     +                                      Mammary Gland RF                                                                            ++         0       0     0                                      Mammary Gland RR                                                                            ++         0       0     0                                      Mandibular LN R                                                                             +++        0       0     0                                      Mandibular LN L                                                                             +++        0       0     0                                      Mediastinal LN                                                                              ++         0       +     0                                      Mesenteric LN +++        0       0     0                                      Parotid LN L  +++        0       0     0                                      Parotid LN R  +++        0       0     0                                      Popliteal LN L                                                                              +          0       0     0                                      Popliteal LN R                                                                              +          0       0     0                                      Prefemoral LN L                                                                             +          0       0     0                                      Prefemoral LN R                                                                             0          0       0     0                                      Prescapular LN L                                                                            0          0       0     +                                      Prescapular LN R                                                                            ++++       0       0     0                                      Renal LN      0          0       0     0                                      Spleen        +++        0       0     0                                      Supramammary LN L                                                                           +++        +       0     0                                      Supramammary LN R                                                                           0          0       0     0                                      Suprapharangeal LN L                                                                        +          0       0     0                                      Suprapharangeal LN R                                                                        0          0       0     0                                      Thymus        0          0       0     0                                      Vagina        +++        0       0     0                                      ______________________________________                                        0 No detectable bacteria by culture of 0.3-1 gm of tissue.                     + Less than 200 colonies/gm tissue.                                           + + More than 300 colonies/gm.                                                +++ More than 1,000 colonies/gm.                                              ++++ More than 100,000 colonies/gm.                                      

Bacteriologic isolations were done on all tissue samples andquantitation of bacteria per gram of tissue were calculated. The resultsfrom four animals--one placebo control and three animals treated withSPLV-entrappedstreptomycin--are presented in Table XV.

10. EXAMPLE: TREATMENT OF SYSTEMIC INFECTIONS

The following examples demonstrate how SPLVs can be used in treatingsystemic infections.

10.1. Effect of Single Treatment of S. Typhimurium Infection UsingSPLV-entrapped Antibiotics

The SPLVs used in the following experiment were prepared by themonophasic solvent system. SPLVs containing gentamicin, nafcillin orboth were prepared as described in Section 6.2.

Ten adult female Swiss Webster mice were infected with S. typhimurium(O.D.₄₂₀ of 0.430 at approximately 5×10⁶ CFU per mouse, I.P., anddivided into 2 groups of 5 mice each. One day post-inoculation with S.typhimurium, groups were treated as follows: Group 1, designatedcontrols, received no treatment; Group 2 received SPLVs (prepared asdescribed in section 6) containing nafcillin-gentamicin in a 1:1 ratio(100 mg/kg body weight) in a total volume of 0.3 ml I.P. (total dose0.27 mg gentamicin per mouse in 0.3 ml and approximately 0.27 mgnafcillin per mouse based upon comparable encapsulation efficiencies fornafcillin and gentamicin). The animals were observed over 14 days forsurvival.

The results of the treatment are as follows: of the controls, after 2days post-inoculation 2 mice survived, after 3 days no survivors wereleft; of Group 2, all animals survived until day 9 post-inoculation whenone animaldied, no other animal died during the 14 day periodpost-inoculation.

The results shown in Table XVI demonstrate the clinical effectiveness ofthe SPLV preparations. There were no survivors in both the control groupand the groups treated with unentrapped antibiotics. However, 100% ofthe infected mice treated with gentamicin and nafcillin entrapped inSPLVs survived.

                  TABLE XVI                                                       ______________________________________                                        EFFECT OF A SINGLE TREATMENT OF                                               S. TYPHIMURIUM INFECTED MICE WITH FREE OR                                     SPLV-ENTRAPPED ANTIBIOTIC                                                               Surviving Animals                                                             Group.sup.a                                                         Day         1            2       3                                            ______________________________________                                        0     (infection)                                                                             10           10    10                                         1     (treatment)                                                                             10           10    10                                         2               3            6     10                                         3               2            5     10                                         4               0            1     10                                         5               0            1     10                                         6               0            0     10                                         7               0            0     10                                         8               0            0     10                                         9               0            0     10                                         14              0            0     10                                         ______________________________________                                         .sup.a Thirty mice divided into 3 groups were infected with S.                typhimurium. The groups were treated as follows: (1) control; (2)             nafcillin/gentamicin; (3) SPLVs containing nafcillin/gentamicin.         

10.2. Effect of Multiple Treatment of S. Typhimurium Infection UsingSPLV-entrapped Antibiotics

The SPLVs used in the following experiment were prepared by themonophasic solvent system method. SPLVs containing chloramphenicol wereprepared as described in Section 6.4.

Twenty adult female Swiss Webster mice were infected with S. typhimurium(O.D.₄₂₀ of 0.430 at approximately 5.5×10⁶, I.P., and divided into 2groups of 10 mice each. One day post-infection and seven dayspost-infection groups were treated as follows: Group 1, designatedcontrols, received no treatment; Group 2 received SPLVs containingchloramphenicol (100 mg/kg body weight) in a total volume of 0.1 ml I.P.The animals were observed over the following 14 day period for survival.

The results shown in Table XVII indicate that 90% of the infectedanimals treated with SPLV-entrapped chloramphenicol survived whereasnone of the untreated animals survived.

These results demonstrate the therapeutic effectiveness of treatment ofsystemic infections with antibiotic-entrapped SPLVs.

                  TABLE XVII                                                      ______________________________________                                        EFFECT OF MULTIPLE TREATMENT MICE                                             INFECTED WITH S. TYPHIMURIUM                                                          Surviving Animals                                                                        Free         SPLV/                                         PAI Day   Controls Chloramphenicol                                                                            Chloramphenicol                               ______________________________________                                        0   (infection)                                                                             10       10         10                                          1   (treatment)                                                                             10       10         10                                          2             3        9          10                                          3             3        6          10                                          4             0        4          10                                          5             0        4          10                                          6             0        1          10                                          7   (treatment)                                                                             0        0           9                                          14            0        0           9                                          ______________________________________                                    

10.3. Enhancement of Antibacterial Activity in Treating SalmonellaTyphimurium Infections Using SPLVs Containing Gentamicin and Nafcillin

In the following example, the antibacterial activity of variouspreparations of the aminoglycoside, gentamicin, and thepencillinase-resistant penicillin, nafcillin, are compared. The resultsdemonstrate that of the preparations tested, treatment of lethalinfections of Salmonella typhimurium (an intracellular infection) inmice is most effective using an SPLV preparation in which bothgentamicin and nafcillin are incorporated into one SPLV preparation.SPLVs containing either gentamicin or nafcillin or both were prepared bythe emulsificationmethod as described in Sections 7.2 and 7.3.

One hundred twenty-five mice were infected by intraperitoneal (I.P.)inoculation of a lethal dose (i.e., 3×10⁶ colony forming units,CFU) ofSalmonella typhimurium in order to establish septicemia. Twenty-fourhours after inoculation the mice were divided into 8 groups ofmice andeach was treated as follows: Group 1 (controls) received no treatment;Group 2 received aqueous nafcillin (100 mg/kg body weight, I.P.); Group3 received aqueous gentamicin (100 mg/kg body weight, I.P.); Group 4received a single preparation containing both aqueous gentamicin (50mg/kg body weight, I.P.) and nafcillin (50 mg/kg body weight, I.P.);Group 5 received SPLVs containing nafcillin (100 mg/kg body weight,I.P.);Group 6 received SPLVs containing gentamicin (100 mg antibiotic/kgbody weight); Group 7 received a mixture of two SPLV preparations, onecontaining gentamicin (50 mg/kg body weight, I.P.) and the other SPLVpreparation containing nafcillin (50 mg/kg body weight, I.P.); and Group8received one SPLV preparation containing both gentamicin (50 mg/kg bodyweight, I.P.) and nafcillin (50 mg/kg body weight, I.P.). Results areshown in Table XVIII.

The results shown in Table XVIII clearly indicate that the SPLVscontainingboth gentamicin and nafcillin were most effective inpreventing mortality due to infection. In fact, the administration ofthe SPLV preparation containing both gentamicin and nafcillin was notonly more effective in preventing mortality than was the administrationof both drugs in an aqueous solution, but surprisingly treatment withthe SPLV preparation containing both gentamicin and nafcillin was moreeffective in preventing mortality than was the simultaneous treatmentwith two populations of SPLVs, one containing gentamicin and the othercontaining nafcillin.

                                      TABLE XVIII                                 __________________________________________________________________________    ENHANCED EFFECT OF SPLV-ENTRAPPED GENTAMICIN AND NAFCILLIN                             SURVIVAL                                                                      DAYS AFTER INFECTION                                                          DAYS POST TREATMENT              %                                   GROUP.sup.a                                                                            1  2  3  4   5   6   7   8   14  SURVIVAL                            __________________________________________________________________________    GROUP 1                                                                       CONTROL        4/25                                                                             0/25                                                                              0/25                                                                              0/25                                                                              0/25                                                                              0/25                                                                              0/25                                                                              0%                                  (untreated)                                                                   GROUP 2                                                                       NAFCILLIN      0/15                                                                             0/15                                                                              0/15                                                                              0/15                                                                              0/15                                                                              0/15                                                                              0/15                                                                              0%                                  (aq.)                                                                         GROUP 3                                                                       GENTAMICIN                                                                             2/15.sup.c                                                                       0/15                                                                             0/15                                                                             0/15                                                                              0/15                                                                              0/15                                                                              0/15                                                                              0/15                                                                              0/15                                                                              0%                                  (aq.)                                                                         GROUP 4                                                                       GENT/NAF    9/10                                                                             5/10                                                                             1/10                                                                              0/10                                                                              0/10                                                                              0/10                                                                              0/10                                                                              0/10                                                                              0%                                  (aq.)                                                                         GROUP 5                                                                       SPLV/             1/15                                                                              1/15                                                                              1/15                                                                              1/15                                                                              0/15                                                                              0/15                                                                              0%                                  NAFCILLIN                                                                     GROUP 6                                                                       SPLV/             1/15                                                                              1/15                                                                              1/15                                                                              1/15                                                                              0/15                                                                              0/15                                                                              0%                                  GENTAMICIN                                                                    GROUP 7                                                                       SPLV/             1/15                                                                              1/15                                                                              1/15                                                                              1/15                                                                              0/15                                                                              0/15                                                                              0%                                  NAFCILLIN                                                                     AND SPLV/                                                                     GENTAMICIN                                                                    GROUP 8                                                                       SPLV/             15/15                                                                             15/15                                                                             15/15                                                                             15/15                                                                             14/15                                                                             14/15                                                                             93%                                 GENT-NAF                                                                      __________________________________________________________________________     .sup.a Each group of mice received a total of 100 mg antibiotic/kg body       weight (except for the control group which received no treatment) 24 hour    after infection with a lethal dose of S. typhimurium (3 × 10.sup.6       CFU, I.P.).                                                                   .sup.b Survival is expressed as the number of mice alive divided by the       total number of mice in the group.                                            .sup.c These mice died immediately after injection of gentamicin due to       acute toxicity of the gentamicin.                                        

10.4. Enhancement of Antibacterial Activity in Treating SalmonellosisUsingSPLVs Containing Gentamicin and Nafcillin

In this example, the antibacterial activity and clinical effectivenessof SPLVs containing both gentamicin and nafcillin are compared to anumber ofother preparations. The results indicate that of thepreparations tested, treatment of Salmonella typhimurium is mosteffective when using an SPLV preparation in which gentamicin andnafcillin are both incorporated into one SPLV preparation.

SPLVs containing no drug and SPLVs containing either gentamicin ornafcillin, were prepared as by the emulsification method described inSection 7.2 using 200 mg EPC and 200 mg of drug. SPLVs containing bothgentamicin and nafcillin were prepared as described in Section 7.3 using200 mg EPC and 200 mg of each drug.

Each SPLV preparation was washed four times and resuspended in thefollowing solutions: (a) SPLVs containing no drug were suspended to atotal volume of 2 ml using physiological saline; (b) SPLVs containingbothnafcillin and gentamicin (SPLV/NAF-GENT) in one liposome preparationwere suspended to a total volume of 2 ml using physiological saline; (c)SPLVs containing nafcillin were suspended to a total volume of 1 mlusing physiological saline. A 0.5 ml aliquot of this suspension wasresuspended to a final volume of 1 ml using physiological saline towhich 20 mg gentamicin was added (SPLV/NAF in gentamicin, aq.); (d)SPLVs containing gentamicin were suspended to a total volume of 1 ml. A0.5 ml aliquot of this suspension was resuspended to a final volume of 1ml using physiological saline to which 20 mg nafcillin was added(SPLV/GENT in nafcillin, aq.); (e) the remaining 0.5 ml aliquot of SPLVscontaining nafcillin in physiologic saline (see (c) above) was added toa 0.5 ml aliquot of SPLVs containing gentamicin in physiologic saline(SPLV/NAF andSPLV/GENT). The resuspended SPLV preparations had thefollowing compositions per 0.1 ml aliquot: (a) SPLVs=20 mg EPC; (b)SPLV/NAF-GENT=20mg EPC, 2 mg nafcillin 2 mg gentamicin; (c) SPLV/NAF ingentamicin, aq.=20 mg EPC, 2 mg nafcillin, 2 mg gentamicin; (d)SPLV/GENT in nafcillin, aq.=20 mg EPC, 2 mg gentamicin, 2 mg nafcillin;and (e) SPLV/NAF and SPLV/GENT=40 mg EPC, 2 mg nafcillin, 2 mggentamicin.

Hilltop mice (20-30 mg each) were infected with Salmonella typhimuriumby intraperitoneal injection of 0.3 ml of a culture of S. typhimurium inBHI broth (Brain Heart Infusion Media, BBL Microbiological Systems,Cockeysville, Md.) grown to an O.D.₄₂₀ of about 0.18.

Twenty seven hours after infection with S. typhimurium the mice weredivided into 7 groups and each group was treated by inoculation of 0.1ml (either I.P. or I.V., intravenous) as follows: Group 1 (controls)were untreated; Group 2 received SPLVs containing no drug (I.V.); Group3 received SPLV/GENT in nafcillin, aq. (100 mg of each antibiotic/kgbody weight, I.V.); Group 4 received SPLV/NAF in gentamicin, aq. (100 mgof each antibiotic/kg body weight, I.V.); Group 5 received a mixture oftwo liposome populations, SPLV/NAF and SPLV/GENT (100 mg of eachantibiotic/kgbody weight, I.V.); Group 6 received SPLV/NAF-GENT (100 mgof each antiobiotic/kg body weight, I.V.); and Group 7 receivedSPLV/NAF-GENT (100mg of each antibiotic per kg body weight, I.P.).Results are shown in TableXIX.

                                      TABLE XIX                                   __________________________________________________________________________    EFFECT ON SPLV ENTRAPPED GENTAMICIN AND                                       NAFCILLIN ON SALMONELLA TYPHIMURIUM INFECTION IN MICE                                    SURVIVAL                                                                      DAYS AFTER TREATMENT        %                                      GROUP      1-3                                                                              4.5                                                                              6  7  8  9  10 11-12                                                                             13 Survival                               __________________________________________________________________________    GROUP 1                                                                       CONTROL    5/5                                                                              3/5                                                                              2/5                                                                              1/5                                                                              1/5                                                                              1/5                                                                              1/5                                                                              0/5 0/5                                                                              0%                                     (untreated)                                                                   GROUP 3                                                                       SPLVS      5/5                                                                              3/5                                                                              3/5                                                                              1/5                                                                              0/5                                                                              0/5                                                                              0/5                                                                              0/5 0/5                                                                              0%                                     (I.V.)                                                                        GROUP 3                                                                       SPLV/GENT  5/5                                                                              3/5                                                                              3/5                                                                              3/5                                                                              2/5                                                                              2/5                                                                              2/5                                                                              1/5 1/5                                                                              20%                                    IN                                                                            NAFCILLIN                                                                     aq. (I.V.)                                                                    GROUP 4                                                                       SPLV/NAF   5/5                                                                              4/5                                                                              4/5                                                                              0/5                                                                              0/5                                                                              0/5                                                                              0/5                                                                              0/5 0/5                                                                              0%                                     IN                                                                            GENTAMICIN                                                                    aq. (I.V.)                                                                    GROUP 5                                                                       SPLV/NAF   5/5                                                                              4/5                                                                              4/5                                                                              3/5                                                                              3/5                                                                              3/5                                                                              1/5                                                                              1/5 0/5                                                                              0%                                     AND SPLV/GENT                                                                 (I.V.)                                                                        GROUP 6                                                                       SPLV/      5/5                                                                              5/5                                                                              5/5                                                                              5/5                                                                              5/5                                                                              4/5                                                                              4/5                                                                              4/5 4/5                                                                              80%                                    NAF-GENT                                                                      (I.V.)                                                                        GROUP 7                                                                       SPLV/      5/5                                                                              5/5                                                                              4/5                                                                              4/5                                                                              4/5                                                                              3/5                                                                              3/5                                                                              3/5 3/5                                                                              60%                                    NAF-GENT                                                                      (I.P.)                                                                        __________________________________________________________________________

These results demonstrate the increased effectiveness of the combinationofnafcillin and gentamicin entrapped in one SPLV preparation in thetreatmentof S. typhimurium infection in vivo whether administeredintravenously or intraperitoneally.

10.5. Enhancement of Antibacterial Activity in Treating SalmonellaTyphimurium Infections Using SPLVs Containing Gentamicin and Nafcillin

In this example, the antibacterial activity and clinical effectivenessof various preparations of the antibiotics gentamicin and nafcillin arecompared. The results indicate that of the preparations tested,treatment of S. typhimurium is most effective when using an SPLVpreparation in which gentamicin and nafcillin are incorporated into oneliposome preparation. SPLVs containing either gentamicin or nafcillin orboth were prepared by the monophasic solvent system process described inSections 6.2 and 6.3.

Sixty-five mice were infected by intraperitoneal (I.P.) inoculation of alethal dose (i.e. 5×10⁶ CFU) of S. typhimurium in order to establishsepticemia. Twenty-four hours after inoculation the mice were dividedinto 3 groups and treated as follows: Group 1 (controls) received notreatment; Group 2 received a single preparation containing both aqueousgentamicin (100 mg/kg body weight) and aqueous nafcillin (100 mg/kg bodyweight, I.P.); Group 3 received one SPLV preparation containingbothgentamicin (100 mg/kg body weight, I.P.) and nafcillin (50 mg/kg bodyweight, I.P.). Results are shown in Table XX.

Results shown in Table XX clearly demonstrate that the SPLVs containinggentamicin and nafcillin coencapsulated were most effective inpreventing mortality due to infection.

                                      TABLE XX                                    __________________________________________________________________________    EFFECT OF SPLV-ENTRAPPED GENTAMICIN AND NAFCILLIN                                    SURVIVAL.sup.b                                                                DAYS AFTER INFECTION            %                                      GROUP.sup.a                                                                          1-2 3   4   5   6   7-10                                                                              11-12                                                                             13-15                                                                             SURVIVAL                               __________________________________________________________________________    GROUP 1                                                                       CONTROL                                                                              20/20                                                                              5/20                                                                              2/20                                                                             0/20                                                                              0/20                                                                              0/20                                                                              0/20                                                                              0/20                                                                              0%                                     (untreated)                                                                   GROUP 2                                                                       GENT/NAF                                                                             20/20                                                                             15/20                                                                             10/20                                                                             6/20                                                                              1/20                                                                              0/20                                                                              0/20                                                                              0/20                                                                              0%                                     (aq.)                                                                         GROUP 3                                                                       SPLV/  25/25                                                                             25/25                                                                             25/25                                                                             25/25                                                                             25/25                                                                             25/25                                                                             24/25                                                                             23/25                                                                             92%                                    GENT-NAF                                                                      __________________________________________________________________________     .sup.a Each animal received a total of 100 mg antibiotic/kg body weight       (except for the control group which received no treatment) 24 hours after     infection with a lethal dose of typhimurium (5 ×10.sup.6 CFU, I.P.)     .sup.b Survival is expressed as the number of mice alive divided by the       total number of mice in the group.                                       

11. EXAMPLE: TREATMENT OF OCULAR AFFLICTIONS

Bacterial and like infections as well as many other afflictions of theeye cause worldwide economic and public health problems, leading, ifuntreatedor improperly treated, to loss of sight and possible death dueto septicemia. Bacterial infections of the eye in animals and man havebeen reported to be caused by a variety of bacteria including but notlimited to: Clostridium spp., Corynebacterium spp., Leptospira spp.,Moraxella spp., Mycobacterium spp., Neisseria spp., Propionibacteriumspp., Proteus spp., Pseudomonas spp., Serratia spps., E. coli spp.,Staphylococcus spp.,Streptococcus spp. and bacteria-like organismsincluding Mycoplasma spp. and Rickettsia spp. Both animals and man serveas reservoirs for potentialspread of infectious bacteria to each other.

Such bacterial infections cannot be treated with antibiotics withoutlengthy and cumbersome treatment schedules resulting in either frequenttreatments, as rapid as every twenty minutes in humans with someinfections, or unacceptably high concentrations of the antibiotic in thetissues. Current treatment methods are difficult for many other reasons.The infectious organism in the surface tissues of the eye in some casesare highly resistant to bactericidal activities of antibiotics, andtopical administration of antibiotics can result in rapid clearing ofthe drug from the eye socket yielding varying contact times. As ageneral rule, treatment of eye infections has to be completely effectivesince anyremaining infection will simply reinfect through lacrimalsecretions and the cycle commences once again. Further, in many casesdrug concentrationsneeded to eliminate the infection can causeimpairment of vision and in certain cases can result in total blindness.The economic impact o±suchdiseases in domestic animals is demonstratedby the millions of dollars which are lost each year since the onlypotential way to combat such infectious diseases is sustained therapyand quarantine.

The following experiments evaluate the effectiveness of treatments usingfree antibiotic in glycerine as compared to antibiotic entrapped inSPLVs for M. bovis infections of the eye.

M. bovis causes infectious keratoconjunctivitis (pink-eye) in cattle.This condition is characterized by blepharospasm, lacrimation,conjunctivitis and varying degrees of corneal opacity and ulceration.Adult cows may develop a mild fever with slightly decreased appetite anda decreased milkproduction. Although a number of antibiotics areeffective against M. bovis, they must be administered early and repeatedoften by topical application or subconjuctival injection.

According to the examples described herein, the effectiveness andduration of action of the therapeutic substance are prolonged. It issurprising that this system is effective with only one or twoadministrations since such infections do not respond to simple ordinarytreatment with antibiotics. The usual treatments often leave smallremaining infections which reinfect the eye so that the infectious cyclewill commence again, unless the infection is completely eradicated bynumerous repetitions of the treatment.

11.1. Treatment of Infectious Keratoconjunctivitis in Mice

C57 black mice (160 mice) were divided into 8 groups. One half of eachgroup was exposed to U.V. irradiation in each eye (in order to createcorneal lesions). All animals were then inoculated with M. bovisinstilledonto the right eye at concentrations of 1×10⁶ bacteria per eye.Twenty-four hours post-inoculation all animals were scored for degree ofcorneal opacity. The eight groups were treated by topical application ofthe following to each eye: Groups 1 and 2 received 10 μl ofSPLV-entrapped streptomycin (30 mg/ml); Groups 3 and 4 received 10 μlstreptomycin (100 mg/ml); Groups 5 and 6 received 10 μl of buffer-filledSPLVs suspended in aqueous streptomycin (100 mg/ml); and Groups 7 and 8received 10 μl of sterile saline (N.B. The uninfected left eyes weretreated with the same topical solutions in order to determine whetherSPLVs would irritate the eye; no irritation was observed). Once daily,animals were scored for progression or regression of corneal lesions andon days 3, 5 and 7 post-treatment right eyes were swabbed and isolationsfor M. bovis were performed on representative animals. M. bovis colonieswere determined by colony morphology and reactivity to flourescentlylabeled antibody to M. bovis pili. Results, shown in Table XXI, revealthat only the SPLV-entrapped streptomycin was effective in eliminatinginfection.

                                      TABLE XXI                                   __________________________________________________________________________    RESULTS OF TREATMENT OF INFECTIOUS KERATOCON-                                 JUNCTIVITIS RESULTING FROM OCULAR INFECTIONS OF                               M. BOVIS IN MICE                                                                        Number of Mice Per Group of 20                                                                     M. Bovis Cultures.sup.a                                  Pre-Treatment                                                                            Post-Treatment                                                                          Days Post-                                               Corneal Opacity.sup.b                                                                    Corneal Opacity.sup.b                                                                   Treatment                                                0 1 2 3  4 0 1 2 3 4 3   5                                          __________________________________________________________________________    Non-radiated Mice                                                             Controls  16                                                                              3 0 1  0 18                                                                              2 0 0 0 4/5 4/5                                        Free Strepto-                                                                           18                                                                              1 1 0  0 18                                                                              2 0 0 0 2/5 2/5                                        mycin.sup.c                                                                   Buffer-filled                                                                           17                                                                              2 1 0  0 18                                                                              1 1 0 0 2/5 3/5                                        SPLVs plus free                                                               Streptomycin.sup.c                                                            SPLVs-Entrapped                                                                         17                                                                              3 0 0  0 20                                                                              0 0 0 0 0/5 0/5                                        Streptomycin.sup.c                                                            UV-Radiated Mice                                                              Controls   1                                                                              1 5 9  4 10                                                                              3 1 2 4 5/5 5/5                                        Free Strepto-                                                                            0                                                                              4 9 7  0 14                                                                              3 2 1 0 3/5 4/5                                        mycin.sup.c                                                                   Buffer-filled                                                                            0                                                                              3 5 10 2 11                                                                              2 4 3 0 3/5 3/5                                        SPLVs plus free                                                               Streptomycin.sup.c                                                            SPLVs-Entrapped                                                                          0                                                                              1 5 11 3 19                                                                              1 0 0 0 0/5 0/5                                        Streptomycin                                                                  __________________________________________________________________________     .sup.a Culture of eyes positive for presence of M. bovis, determined by       fluorescent antibody staining.                                                .sup.b Scoring of normal cornea; 1 = loss of normal luster; 2 = small foc    of opacity; 3 = partial opacity of cornea; 4 = total opacity of cornea.        .sup.c Total administration 10 μl (1.0 mg streptomycin per eye).      

11.2. Treatment of Rabbit Conjunctiva Using SPLV-entrapped Antibiotic

M. bovis, ATCC strain 10900, were diluted to a concentration of 1×10⁷cells per ml in sterile saline (0.085% NaCl). Aliquots (0.1 ml) ofbacterial suspensions were inoculated topically into the eyes of tenadult female rabbits. Samples for cultures were taken daily by swabbingthe conjunctivae and plated onto blood agar plates for isolation of M.bovis. Three days post-inoculation, rabbits were divided into 3 groups:2 animals (controls) received no treatment; 4 animals receivedstreptomycin in sterile saline (concentration 10 mg/kg body weight); and4animals received SPLV-entrapped streptomycin in a sterile salinesolution (concentration 10 mg streptomycin/kg body weight). Allsolutions were administered topically into each eye. After 24 hours, theswabbing of conjunctivae of all rabbits was resumed and continued dailyfor seven days. The results of isolation for M. bovis on blood agarplates are shownin Table XXII.

                  TABLE XXII                                                      ______________________________________                                        RESULTS OF ISOLATION FROM RABBIT                                              CONJUNCTIVAE AFTER TOPICALLY INFECTING                                        WITH M. BOVIS AND TREATING WITH AQUEOUS                                       OR SPLV-ENCAPSULATED STREPTOMYCIN                                                          M. bovis Cultures.sup.a                                                       Days Post-Infection                                                      Animal Pre-Treatment.sup.b                                                                       Post-Treatment.sup.c                               Group     Number   1       2     3   4   5   6   7                            ______________________________________                                        Control   1        0       +     +   +   +   +   +                                      2        0       +     +   +   +   +   +                            Streptomycin.sup.d                                                                      1        0       +     +   +   +   +   +                                      2        0       0     +   +   +   +   +                                      3        0       +     +   +   +   +   +                                      4        0       +     +   +   +   +   +                            SPLV-     1        0       0     +   0   0   0   0                            Entrapped 2        0       +     +   0   0   0   0                            Streptomycin.sup.e                                                                      3        0       +     +   0   0   0   0                                      4        0       +     +   0   0   0   0                            ______________________________________                                         .sup.a Cultures scored for presence of M. bovis colonies on blood agar        plates after 24 hours at 37° C. Plus (+) represents greater than o    equal to 1 CFU M. bovis per isolate; 0 represents no detectable colonies.      .sup.b All animals inoculated with 1 × 106 CFU M. bovis topically i    each eye.                                                                      .sup.c Animals treated with 0.1 ml solution topically in each eye.            .sup.d Streptomycin (10 mg/kg body weight) in sterile saline solution.        .sup.e SPLVentrapped streptomycin (10 mg/kg body weight) in sterile salin    solution.                                                                 

11.3. Treatment of Keratoconjunctivitis Resulting From SubcutaneousInfections

M. bovis, ATCC strain 10900, were diluted to a concentration of 1×10⁷cells per ml in sterile saline. Aliquots (0.1 ml) of bacterialsuspensions were inoculated into the eyes of adult rabbits whichhad beenpreviously infected as described in Section 9.2. and were not treatedwith SPLVs. The right eyes of all nine rabbits were inoculated with 0.1ml of M. bovis subcutaneously into conjunctival tissue and in thelefteyes of all rabbits were inoculated with 0.1 ml of M. bovistopically.Cultures were taken daily from conjunctivae of both eyes fromall rabbits and plated onto blood agar plates for isolation of M. bovis.Three days post-inoculation, rabbits were divided into 3 groups: 2animals received no treatment; 3 animals received streptomycin in astandard ophthalmic glycerin suspension (concentration of streptomycin10 mg/kg body weight); and 4 animals received a saline suspension ofSPLV-entrapped streptomycin (10 mg of streptomycin sulfate per kg ofbody weight). The suspension or solution was administered topically (0.1ml) into each eye. After 24 hoursand on each of the next five days,conjunctival swabbings were taken from all rabbits. The results ofisolation for M. bovis on blood agar plates are shown in Table XXIII.Necropsies were performed on all animals at the termination ofexperiments and conjunctivae were removed from all animals.These werescored for vascularization, and were minced, homogenized and plated ontoblood agar plates for isolation of M. bovis. Results are shownin TableXXIV.

                  TABLE XXIII                                                     ______________________________________                                        RESULTS OF ISOLATION FROM RABBIT CON-                                         JUNCTIVAE AFTER INOCULATION OF M. BOVIS                                       INTO CONJUNCTIVAL MEMBRANES AND TREAT-                                        MENT WITH STREPTOMYCIN IN OPHTHALMIC                                          GLYCERINE SOLUTION OR SPLV-ENCAPSULATED                                       STREPTOMYCIN IN SALINE                                                                    M. bovis Cultures.sup.a                                                       Days Post Infection.sup.c                                                Animal Pre-treatment                                                                             Post-treatment                                      Group    Number.sup.b                                                                           1       2     3     4    5                                  ______________________________________                                        Control  1        +       +     +     +    +                                           2        +       +     +     +    +                                  Streptomycin                                                                           1        +       +     +     +    +                                  in Glycerine                                                                           2        +       +     +     +    +                                  solution.sup.d                                                                         3        +       +     +     +    +                                  SPLV-    1        +       +     0     0    0                                  Encapsulated                                                                           2        +       +     0     0    0                                  Streptomycin.sup.e                                                                     3        +       +     0     0    0                                           4        +       +     0     0    0                                  ______________________________________                                         .sup.a Cultures scored for presence of M. bovis colonies on blood agar        plates after 24 hours at 37°  C. Plus (+) represents greater than      or equal to 1 CFU M. bovis per isolate; 0 represents no detectable            colonies.                                                                     .sup.b All animals were inoculated with 1 × 10.sup.6 CFU M. bovis       topically in both eyes; 1 × 10.sup.6 CFU M. bovis was injected into     conjunctival membranes, in right eyes; and 1 × 10.sup.6 CFU M. bovi    was applied topically in left eyes.                                            .sup.c Animals treated with 0.1 ml solution topically in each eye.            .sup.d Animals treated topically in each eye with streptomycin (10 mg/kg      body weight) in opthalmic glycerine base.                                     .sup.e Animals treated topically in each eye with SPLVencapsulated            streptomycin (10 mg/kg body weight) in sterile saline solution.          

                  TABLE XXIV                                                      ______________________________________                                        RESULTS FROM NECROPSY OF THE ORBIT AND                                        ASSOCIATED TISSUES FROM RABBITS AFTER                                         INOCULATION WITH M. BOVIS INTO CONJUNCTIVAL                                   TISSUES AND TREATMENT WITH EITHER STREPTO-                                    MYCIN IN OPHTHALMIC GLYCERINE SOLUTION                                        OR SPLV-ENCAPSULATED STREPTOMYCIN IN                                          STERILE SALINE.sup.a                                                                        Isolation                                                                     of M. bovis                                                                           Vascularization                                                       Cultures                                                                              of Right Eye.sup.b                                      ______________________________________                                        Control                                                                       A               +         2+                                                  B               +         2+                                                  Streptomycin in                                                               Glycerine Solution                                                            A               +         2+                                                  B               +         1+                                                  C               +         2+                                                  D               +         2+                                                  SPLV-encapsulated                                                             Streptomycin                                                                  A               0         0                                                   B               0         0                                                   C               0         0                                                   D               0         0                                                   ______________________________________                                         .sup.a Legends are same as Table XXIII, performed on day 5, post              infection.                                                                    .sup.b Vascularization scored as follows: 0 = vessels normal; 1 = some        vessels definitely dilated and infiltrated by minor vessels; 2 = diffuse      red with individual vessels not easily discernible; 3 = diffuse beefy red    vascular leakage and effusion of blood into conjunctivae.                 

11.4. Evaluation of the Effectiveness of SPLVS as Compared to LiposomePreparations in the Treatment of Ocular Infections

M. bovis (ATCC strain 10900) were diluted to a concentration of 1×10⁷cells per ml in sterile saline. Aliquots (0.1 ml) of bacterialsuspensions were inoculated subcutaneously into the conjunctivaltissuesof both eyes in adult rabbits. Swabbings were taken daily fromconjunctivae of both eyes from all rabbits and plated onto blood agarplates for isolation of M. bovis. Five days post-inoculation, rabbitsweredivided into 6 groups: 2 animals received no treatment (controls); 3animals received a suspension of SPLV-encapsulated streptomycin (10 mgof streptomycin sulfate per kg of body weight) which when diluted 1:100had an O.D.₄₈₀ (optical density at 480 nm) equal to 0.928; 3 animalsreceived a suspension of SPLV-encapsulated streptomycin (10 mg ofstreptomycin sulfate per kg of body weight) which when diluted 1:100 hadan O.D.₄₈₀ equal to 0.449; 3 animals received a suspension ofSPLV-encapsulated streptomycin (10 mg streptomycin sulfate per kg ofbody weight) which when diluted 1:100 had an O.D.₄₈₀ equal to 0.242; 3animals received a suspension of SPLV-encapsulated streptomycin (10 mgstreptomycin sulfate per kg body weight) which when diluted 1:100 had anO.D.₄₈₀ equal to 0.119: and 2 animals received a suspension ofmultilamellar vesicles (MLVs) containing streptomycin (10 mgstreptomycin sulfate per kg of body weight) with an O.D.₄₈₀ of a 1:100dilution equal to 0.940. MLVS were made by the process of Fountain etal. Curr. Micro. 6:373 (1981) by adding streptomycin sulfate to thedried lipid filmwhich was then vortexed, and allowed to swell for twohours; the non-entrapped streptomycin was removed by repeatedcentrifugation.

                                      TABLE XXV                                   __________________________________________________________________________    ISOLATION OF M. BOVIS FROM INFECTED RABBIT CONJUNCTIVAE                       AFTER TREATMENT WITH DILUTIONS OF SPLV-ENCAPSULATED                           STREPTOMYCIN IN SALINE OR MLV-ENCAPSULATED STREPTO-                           MYCIN IN SALINE                                                                           Isolation of M. bovis.sup.a                                                   Days Post-Infection                                                      Animal                                                                             Pre-Treatment Post-Treatment                                      Group  Number                                                                             1 2 3 4 5 6 7 8 9 10                                                                              11                                                                              12                                                                              13                                                                              14                                      __________________________________________________________________________    Control                                                                              1    + + + + + + + + + + + + + +                                              2    + + + + + + + + + + + + + +                                       MLV-                                                                          encapsulated                                                                         1    + + + + + + + 0 0 + + + 0 +                                       Streptomycin                                                                         2    + + + + + + 0 + + 0 0 + + 0                                       SPLV-                                                                         encapsulated                                                                         1    + + + + + 0 0 0 0 0 0 0 0 0                                       Streptomycin                                                                         2    + + + + + 0 0 0 0 0 0 0 0 0                                       (undiluted)                                                                          3    + + + + + 0 0 0 0 0 0 0 0 0                                       SPLV-                                                                         encapsulated                                                                         1    + + + + + + 0 + + + + + + +                                       Streptomycin                                                                         2    + + + + + 0 0 + 0 0 0 0 0 0                                       (1:2 dilution)                                                                       3    + + + + + 0 0 0 0 + 0 0 0 0                                       SPLV-                                                                         encapsulated                                                                         1    + + + + + + + 0 0 0 0 0 0 0                                       Streptomycin                                                                         2    + + + + + + 0 0 0 + 0 0 0 0                                       (1:4 dilution)                                                                       3    + + + + + 0 0 + + + + + + +                                       SPLV-                                                                         encapsulated                                                                         1    + + + + + 0 0 + + + + + + +                                       Streptomycin                                                                         2    + + + + + 0 0 0 0 0 0 0 0 0                                       (1:6 dilution)                                                                       3    + + + + + + + + 0 + + + + +                                       __________________________________________________________________________     .sup.a All animals inoculated with 1 × 10.sup.6 CFU M. bovis by         injection into conjunctival membranes of both eyes. Conjunctival swabbing    were plated on blood agar. Cultures scored for presence of M. bovis            colonies on blood agar plates after 24 hours at 37° C.; + = greate    than or equal to 1 CFU; 0 = no detectable cultures.                       

                  TABLE XXVI                                                      ______________________________________                                        RESULTS FROM NECROPSY OF THE ORBIT AND                                        ASSOCIATED TISSUES FROM RABBITS AFTER                                         INOCULATION WITH M. BOVIS INTO CONJUNC-                                       TIVAL TISSUES AND TREATMENT WITH EITHER                                       MLV-ENCAPSULATED STREPTOMYCIN, SPLV-                                          ENCAPSULATED STREPTOMYCIN OR DILUTIONS                                        OF SPLV-ENCAPSULATED STREPTOMYCIN.sup.a                                                    Isolation Vascularization                                                                           Lacrimal                                          Animal                                                                              of M. Bovis                                                                             of Eyes.sup.b                                                                             Discharge.sup.c                            ______________________________________                                        Control  1       +         1+        1+                                                2       +         1+        1+                                       MLV-                                                                          encapsulated                                                                           1       +         1+        1+                                       Streptomycin                                                                           2       0         1+        0                                        SPLV-                                                                         encapsulated                                                                           1       0         0         0                                        Streptomycin                                                                           2       0         1+        0                                        (undiluted)                                                                            3       0         0         0                                        SPLV-                                                                         encapsulated                                                                           1       +         2+        2+                                       Streptomycin                                                                           2       0         0         0                                        (1:2 dilution)                                                                         3       0         1+        0                                        SPLV-                                                                         encapsulated                                                                           1       0         0         0                                        Streptomycin                                                                           2       0         1+        0                                        (1:4 dilution)                                                                         3       +         1+        0                                        SPLV-                                                                         encapsulated                                                                           1       +         1+        1+                                       Streptomycin                                                                           2       0         1+        0                                        (1:6 dilution)                                                                         3       +         1+        0                                        ______________________________________                                         .sup.a Legends are same as Table XXV, performed on day 14 postinfection.      .sup. b Vascularization scored as follows: 0 = vessels normal; 1 = some       vessels dilated and infiltrated by minor vessels; 2 = diffuse red with        individual vessels not easily discernable; 3 = diffuse beefy red, vascula    leakage and effusion of blood into conjunctivae.                               .sup.c Discharge scored as follows: 0 = no discharge; 1 = discharge with      moistening of lids and hairs adjacent to lids; 2 = discharge with             moistening of lids, hairs and areas adjacent to eyes.                    

The suspensions were administered topically into each eye. After 24hours, conjunctival swabbings were taken from all rabbits daily for 9days and plated onto blood agar. The results of isolation for M. bovison blood agar plates are shown in Table XXV. Necropsies were performedon all animals. These were scored for lacrimal secretions, andconjunctivae were removed aseptically from all animals. These werescored for vascularization, and were minced, homogenized and plated ontoblood agar plates for isolation of M. bovis. Results are shown in TableXXVI.

12. EXAMPLE: TREATMENT OF VIRAL INFECTIONS

Lymphocytic choriomeningitis virus (LCMV), a member of the Arenavirusgroup, is known to cause diseases in man and LCMV infection is fatal inmice inoculated intracerebrally with this virus. The death of mice iscaused by the immune cells which react against virus-infected cells. Thevirus does not kill the cells in which it multiplies, therefore, thetherapeutic agent used in mice must either inhibit virus multiplicationsothat the immune cells will not be activated, and/or inhibit theactivation of immune cells.

The following example demonstrates the effectiveness of treating viralinfections by administering a SPLV-encapsulated antiviral compound.

12.1. TREATMENT OF LETHAL LYMPHOCYTIC CHORIOMENINGITIS VIRUS INFECTIONSIN MICE

Swiss mice 2 months of age were inoculated intracerebrally with a lethaldose of LCM virus, i.e., 100 plaque forming units (PFU) in 0.05 mlinoculum per mouse. Mice were divided into 4 groups of 7 animals eachand were treated on days 2, 3 and 4 post-infection by intraperitonealinjections with 0.1 ml/dose/mouse as follows: (1) the "SPLV-R group" wastreated with a suspension of egg phosphatidylcholine SPLVs containing 3mgRibavarin/ml. SPLVs were prepared using 100 mg lipids and 0.3 ml of100 mg drug/ml in PBS buffer; the entrapment of drug was 10%; (2) the"R-group" was treated with a solution of Ribavarin 3 mg/ml in PBS; (3)the "SPLV-group" was treated with buffer-filled SPLVs (i.e., SPLVsprepared asabove but without Ribavarin); and (4) the "control group" wastreated with PBS. On day 5 post-infection 2 mice from each group weresacrificed and their spleens homogenized (2 spleens/group werehomogenized in PBS at 1/20weight per volume buffer). The plaque formingunits (PFU) per ml were determined for each suspension. The remaining 5mice in each groups were observed for lethality two times daily for 30days. The results are presented in Table XXVII.

                  TABLE XXVII                                                     ______________________________________                                        TREATMENT OF LETHAL LCM VIRUS INFECTION                                       IN MICE.sup.a                                                                                       Virus Recovered from Spleen                             Group      Lethality.sup.c                                                                          (PFU × 10.sup.5 /ml).sup.c                        ______________________________________                                        Control    5/5        7.0                                                     SPLV-group 5/5        6.9                                                     R-group    5/5        5.2                                                     SPLV-R-Group                                                                             3/5        3.4                                                     ______________________________________                                         .sup.a Two month old mice were each inoculated intracerebrally with a         lethal dose, i.e., 100 PFU of LCM virus in 0.05 ml inocula.                   .sup.b Lethality is expressed as number dead/number in group.                 .sup.c On the fifth day postinfection 2 mice from each group were             sacrificed and their spleens homogenized at a concentration of 1 gm           spleen/20 ml homogenate.                                                 

Table XXVII clearly indicates a decrease in lethality and a decrease inthevirus recoverable from the infected animals. We have not yetdetermined whether these results are due to the anti-viral activity ofthe ribavarin which is released from the SPLVs or whether it is due toan immunomodulation of the mouse host during the sustained release ofribavarin from the SPLVs.

13. EXAMPLE: ENHANCEMENT OF ANTIBACTERIAL ACTIVITY IN TREATINGCORYNEBACTERIUM RENALE PYELONEPHRITIS USING SPLVS CONTAINING GENTAMICINAND NAFCILLIN

In this example, the antibacterial activity and clinical effectivenessof various preparations of gentamicin and nafcillin are compared. Theresultsindicate that of the preparations tested, treatment ofCorynebacterium renale pyelonephritis is most effective when using anSPLV preperation in which gentamicin and nafcillin are both incorporatedinto one liposome preparation.

13.1. Preparation of SPLVS

The SPLVs containing either gentamicin or nafcillin or both wereprepared as described in Section 6.2.

13.2. Infection of Mice Using Corynebacterium Renale

A Corynebacterium renale pyelonephritis was induced in adult Hilltopmice (20-30 gm each) essentially by the method of Shumono and Yanagawa(Infection and Immunity, April 1977, pp. 263-67) as follows: each mousewas anesthetized using ether, the abdominal wall was incised and thebladder isolated. The bladder contents were evacuated by applying gentlepressure. A suspension of C. renale in BHI (Blood, Heart Infusionmedium, BBL Microbiological Systems, Cockeysville, Md.) at aconcentration of 10⁷ CFU (colony forming units) per ml was inoculatedinto the bladderuntil full (approximately 0.1 to 0.2 ml per injection or10⁶ organismsper mouse bladder). The abdominal wall was then closed. TheC. renale had been prepared by growing C. renale ATCC strain No. 10848overnight in BHI broth. Organisms were then suspended in saline to anO.D.₄₂₀ of approximately 0.78. Serial dilutions were plated on agar inorder to determine the CFU per ml for each dilution.

13.3 Treatment of Infected Mice

Twenty four hours after inoculation with C. renale the mice were dividedinto 7 groups and each group was treated as follows: Group 1 (controls)received no treatment; Group 2 received aqueous gentamicin (100 mg/kgbodyweight, I.P.); Group 3 received SPLVs containing gentamicin (100mg/kg bodyweight, I.P.); Group 4 received aqueous nafcillin (100 mg/kgbody weight, I.P.): Group 5 received SPLVs containing nafcillin (100mg/kg body weight,I.P.); Group 6 received a single aqueous preparationcontaining both gentamicin (100 mg/kg body weight) and nafcillin (100mg/kg body weight) I.P.; and Group 7 received one SPLV preparationcontaining both gentamicin(100 mg/kg body weight) and nafcillin (100mg/kg body weight) I.P. Results are shown in Table XXVIII.

                                      TABLE XXVIII                                __________________________________________________________________________    EFFECT OF SPLV ENTRAPPED GENTAMYCIN AND                                       NAFCILLIN ON C. RENALE PYELONEPHRITIS IN MICE                                          SURVIVAL                                                                      DAYS AFTER TREATMENT        %                                        GROUP.sup.a                                                                            1   2   3   4   5   6   7   SURVIVAL                                 __________________________________________________________________________    GROUP 1                                                                       CONTROLS 15/15                                                                             15/15                                                                             7/15                                                                              1/15                                                                              1/15                                                                              0/15                                                                              0/15                                                                              0                                        (untreated)                                                                   GROUP 2                                                                       GENTAMYCIN                                                                             10/10                                                                             10/10                                                                             4/10                                                                              1/10                                                                              10/10                                                                             1/10                                                                              1/10                                                                              10                                       (100 mg/kg)                                                                   GROUP 3                                                                       SPLV-GENT                                                                              10/10                                                                             10/10                                                                             10/10                                                                             5/10                                                                              5/10                                                                              3/10                                                                              0/10                                                                              0                                        (100 mg/kg)                                                                   GROUP 4                                                                       NAFCILLIN                                                                              10/10                                                                             10/10                                                                             7/10                                                                              0/10                                                                              0/10                                                                              0/10                                                                              0/10                                                                              0                                        (100 mg/kg)                                                                   GROUP 5                                                                       SPLV-NAF 10/10                                                                             10/10                                                                             5/10                                                                              0/10                                                                              0/10                                                                              0/10                                                                              0/10                                                                              0                                        (100 mg/kg)                                                                   GROUP 6                                                                       (aq.)                                                                         GENT/NAF 10/10                                                                             10/10                                                                             8/10                                                                              0/10                                                                              0/10                                                                              0/10                                                                              0/10                                                                              0                                        (100 mg/kg                                                                    each)                                                                         GROUP 7                                                                       SPLV-    10/10                                                                             10/10                                                                             10/10                                                                             10/10                                                                             10/10                                                                             10/10                                                                             10/10                                                                             100                                      GENT/NAF                                                                      (100 mg/kg                                                                    each)                                                                         __________________________________________________________________________     .sup.a All mice were treated by intraperitoneal injection 24 hours after      infection.                                                               

The results in Table XXVII clearly indicate that the SPLVs containingboth gentamicin and nafcillin were most effective in preventingmortality due to C. renale pyelonephritis.

In another set of experiments, the effectiveness of gentamicin andnafcillin entrapped in one SPLV preperation was also compared to theeffectiveness of administering the two drugs separately contained inSPLVs. Accordingly, mice were infected with C. renale as described inSection 13.2. Twenty-four hours after inoculation with C. renale themice were divided into 4 groups and each group was treated as follows:Group 1 (control) received no treatment; Group 2 received aqueousnafcillin (100 mg/kg body weight, I.P.) followed by aqueous gentamicin(100 mg/kg body weight, I.P.) administered 1 hour after the nafcillin(NAF-GENT, aq.; N.B., the aqueous preparations of nafcillin andgentamicin were administered one hour apart in order to prevent in situinactivation of the drugs); Group 3 received a mixture of two SPLVpreparations, one containing gentamicin (SPLV-GENT; 100 mg/kg bodyweight) and the other SPLV preparation containing nafcillin (SPLV-NAF;100 mg/kg body weight) I.P.; and Group 4 received a SPLV preparation(SPLV/GENT-NAF) containing both gentamicin (100 mg/kg body weight) andnafcillin (100 mg/kg body weight) I.P. The results shown in Table XXIXdemonstrate that the SPLV/GENT-NAF preparation was the most effective intreating the infection.

                                      TABLE XXIX                                  __________________________________________________________________________    EFFECT OF SPLV ENTRAPPED GENTAMICIN AND                                       NAFCILLIN ON C. RENALE PYELONEPHRITIS IN MICE                                        SURVIVAL                                                                      DAYS AFTER TREATMENT     %                                             GROUP.sup.a                                                                          1   2   3  4   5  6-11                                                                             12-14                                                                             SURVIVAL                                      __________________________________________________________________________    GROUP 1                                                                       CONTROLS                                                                             7/8 2/8 1/8                                                                              0/8 0/8                                                                              0/8                                                                              0/8 0                                             GROUP 2                                                                       NAF-GENT                                                                             10/10                                                                             10/10                                                                             1/10                                                                             0/10                                                                              0/10                                                                             0/10                                                                             0/10                                                                              0                                             (aq.)                                                                         GROUP 3                                                                       SPLV-GENT                                                                            10/10                                                                             10/10                                                                             6/10                                                                             3/10                                                                              2/10                                                                             0/10                                                                             0/10                                                                              0                                             SPLV-NAF                                                                      GROUP 4                                                                       SPLV/   9/10                                                                              9/10                                                                             9/10                                                                             9/10                                                                              9/10                                                                             9/10                                                                             8/10                                                                              80                                            GENT-NAF                                                                      __________________________________________________________________________     .sup.a All mice were treated by intraperitoneal injection 24 hours after      infection.                                                               

The surviving mice which were treated with the SPLV preparationcontaining both gentamicin and nafcillin were sacrificed at day 14 andthe right kidneys were tested for the presence of C. renale whereas theleft kidneyswere analyzed histologically.

The right kidneys were homogenized in BHI media. The homogenate wasserially diluted and plated on agar. No growth of organisms was detectedin cultures of the right kidneys of the 8 surviving mice. Histologicexamination of the left kidney revealed no lesions in 5/8 of the kidneyssampled, minimal to moderate chronic inflammation in the lining of thepelvis of 2 mice, and purulent pyelonephritis with focal necrosis andacute purulent inflammatory reaction in the center left kidney of only 1mouse. Thus, histologic and bacteriological cure was demonstrated in thesurviving animals.

14. EXAMPLE: ENHANCEMENT OF ANTIBACTERIAL ACTIVITY IN TREATINGPSEUDOMONAS AERUGINOSA PYELONEPHRITIS USING SPLVS CONTAINING TOBRAMYCINAND TICARCILLIN

In this example, the antibacterial activity and clinical effectivenessof various preparations of tobramycin (an aminoglycoside antibiotic) andticarcillin (a β-lactam antibiotic) are compared. The results indicatethat of the preparations tested, treatment of Pseudomonas aeruginosapyelonephritis is most effective when using an SPLV preparationin whichtobramycin and ticarcillin are both incorporated into oneliposomepreparation.

SPLVs containing both tobramycin and ticarcillin were prepared by themonophasic solvent system method as follows: a 10 ml ethanol solution of100 mg EPC was prepared in a round bottom flask. Then 100 mg ticarcillinin 1.5 ml PBS was added to the EPC ethanol solution to which 100 mgtobramycin in 0.5 ml PBS lacking divalent cations (PBS⁻) was added. Theresulting mixture (a dispersion) was evaporated at 54° C. for 3minutesuntil a film formed on the side of the vessel. Then 10 ml of PBS wasadded and the mixture was agitated to form and resuspend the SPLVs.

SPLVs containing either tobramycin or ticarcillin were prepared asdescribed above except that 100 mg of either tobramycin or 100 mg ofticarcillin in PBS was added to the EPC ethanol solution.

14.1. Treatment of Infected Rats

Sprague Dawley rats (approximately 0.2 kg each) were infected with P.aeruginosa by the following technique: female rats were anesthetizedusingBrevital (10.64 mg/200 gm rat) administered subcutaneously. Theurinary bladder was exposed by a midline incision made after shaving theabdomen. A small incision was made in the bladder and all urine wasdrained after which a zinc pellet (3 mm in diameter) was inserted intothe bladder. The bladder incision was tied off using size 000 blackbraided Type B silk thread and a 0.1 ml inoculum of P. aeruginosaculture which was grown overnight in TSB (Trypticase Soy Broth, BBLMicrobiological Systems, Cockeysville, Md.) was injected into thebladder. The abdominal incision was then closed using stainless steelclamps on the abdominal muscle and on the skin.

Infected rats were divided into 5 groups which were treated with twodoses of the following preparations administered intraperitoneally at 4and 28 hours after inoculation with P. aeruginosa: Group 1 (controls)received notreatment; Group 2 received aqueous tobramycin (4 mg/kg bodyweight); Group3 received SPLVs containing tobramycin (4 mg/kg bodyweight); Group 4 received aqueous tobramycin (400 mg/kg body weight);and Group 5 received one SPLV preparation (MPV/TIC-TOBRA) containingboth tobramycin (4 mg/kg body weight) and ticarcillin (4 mg/kg bodyweight).

The surviving rats were sacrificed at day 6 and each pair of kidneys wastested for the presence of P. aeruginosa as follows: after each kidneywasremoved, it was placed on a petri dish containing ethanol, flamed andthen homogenized in 2 ml TSB. The homogenate was adjusted to finalvolume of 10ml using TSB. Serial 10-fold dilutions of the homogenatewere plated in duplicate on agar, and the CFU/ml were determined foreach pair of kidneys. Results are shown in Table XXX.

                                      TABLE XXX                                   __________________________________________________________________________    EFFECT ON SPLV ENTRAPPED TOBRAMYCIN AND                                       TICARCILLIN ON P. AERUGINOSA PYELONEPHRITIS IN RATS                                              LOG.sub.10 CFU OF P. AERUGINOSA                                       SURVIVORS                                                                             RECOVERED IN KIDNEY                                                   6 DAYS POST                                                                           HOMOGENATE RAT                                             GROUP      INFECTION                                                                             1  2  3 4  5  6  7                                         __________________________________________________________________________    GROUP 1                                                                       CONTROLS   5/7     6  4  4 5  8  ND ND                                        (untreated)                                                                   GROUP 2                                                                       TOBRAMYCIN 7/7     4  3  4 0  0  4  0                                         (aq.)                                                                         (4 mg/kg)                                                                     GROUP 3                                                                       SPLV-TOBRA 4/7     3  7  4 0  ND ND ND                                        (4 mg/kg)                                                                     GROUP 4                                                                       TICARCILLIN                                                                              5/7     2  5  4 0  0  ND ND                                        TOBRAMYCIN (aq.)                                                              (4 mg/kg-400                                                                  mg/kg)                                                                        GROUP 5                                                                       SPLV/TIC-TOBRA                                                                           7/7     0  0  0 0  0  4  0                                         (400 mg/kg-4                                                                  mg/kg)                                                                        __________________________________________________________________________

These results indicate that the combination of tobramycin andticarcillin contained in one SPLV preparation was most effective in thetreatment of Pseudomonas pyelonephritis.

14.2. Effects of Different Treatments of Infected Rats

Female Sprague-Dawley rats were infected with P. aeruginosa as describedabove, and treated with various SPLV-drug combinations as listed inTablesXXXI to XXXIV. The animals were observed daily for the 6 daytreatment period and any dead animals were removed from the cages andrecorded. Fourhours after the final treatment on day 6, the animals weresacrificed. Bothkidneys were removed by aseptic technique placed on apetri dish containingethanol flamed and then homogenized in 2 ml TSB.Each pair of kidneys was homogenized in 4 ml TSB and then diluted with 4ml of media (10⁻¹ dilution). TS (Trypticase Soy) agar plate counts ofthe homogenized kidneys were made for each animal and compared with theplate counts made on the diluent treated control group. Dilutions foruntreated controls: 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷. Dilutionsfor treated kidney homogenates: 10⁻¹, 10⁻²,10⁻³, 10⁻ 4, 10⁻⁵, 10⁻⁶,10⁻⁷. Results are presented in Tables XXXI to XXXIV, below.

                                      TABLE XXXI                                  __________________________________________________________________________    EFFECT OF SPLV-ENTRAPPED TOBRAMYCIN ADMINISTERED INTRA-                       PERITONEALLY ON P. AERUGINOSA PYELONEPHRITIS IN RATS                                                                MEAN                                    EXPERI-   I.P..sup.a         #        TITER OF                                                                             %                                MENTAL    PER DAY                                                                             # ANIMALS                                                                            #     CLEARED OF                                                                             INFECTED                                                                             CLEARED OF                       GROUP     (mg/kg)                                                                             IN GROUP                                                                             DEATHS                                                                              INFECTION                                                                              ANIMALS                                                                              INFECTION                        __________________________________________________________________________    CONTROLS  --    110    18    4        10.sup.5 -10.sup.6                                                                    4.3                             (UNINFECTED/                                                                  UNTREATED)                                                                    FREE      4     16     2     4        10.sup.4 -10.sup.5                                                                   28.6                             TOBRAMYCIN                                                                              2      7     2     0        10.sup.4 -10.sup.5                                                                   0                                          1      7     1     1        10.sup.5 -10.sup.6                                                                   17.0                                         0.5  7     0     0        10.sup.5 -10.sup.6                                                                   0                                            0.25                                                                               7     1     1        10.sup.4 -10.sup.5                                                                   13.8                             SPLV-     4     40     3     37        0     100.00                           TOBRAMYCIN                                                                              2     28     2     25        0     96.1                                       1     35     5     23       10.sup.1                                                                             65.7                                         0.5 14     3     7        10.sup.4                                                                             70.0                                         0.25                                                                              15     2     8        10.sup. 5 -10.sup.7                                                                  61.5                             __________________________________________________________________________     .sup.(a) Six daily intraperitoneal doses.                                

Table XXXI clearly shows the superiority of SPLV entrapped tobramycin inclearing infection due to P. aeruginosa in a single daily dose regimen.The SPLV-entrapped tobramycin was 10 times more effective than free drugalone.

                                      TABLE XXXII                                 __________________________________________________________________________    EFFECT OF SPLV-ENTRAPPED TICARCILLIN ADMINISTERED INTRA-                      PERITONEALLY ON P. AERUGINOSA PYELONEPHRITIS IN RATS                                                                MEAN                                    EXPERI-   I.P..sup.a         #        TITER OF                                                                             %                                MENTAL    PER DAY                                                                             # ANIMALS                                                                            #     CLEARED OF                                                                             INFECTED                                                                             CLEARED OF                       GROUP     (mg/kg)                                                                             IN GROUP                                                                             DEATHS                                                                              INFECTION                                                                              ANIMALS                                                                              INFECTION                        __________________________________________________________________________    CONTROLS  --    110    18    4        10.sup.5 -10.sup.6                                                                    4.3                             (UNINFECTED/                                                                  UNTREATED)                                                                    FREE      400   9      0     8        10.sup.6                                                                             89.0                             TICARCILLIN                                                                             200   7      1     0        10.sup.4 -10.sup.5                                                                   0                                          100   7      2     1        10.sup.5 -10.sup.6                                                                   16.6                                       50    7      0     1        10.sup.4 -10.sup.5                                                                   14.3                             SPLV-     50    9      1     8         0     100.00                           TICARCILLIN                                                                             25    27     5     22        0     100.00                                       12.5                                                                              28     0     26       10.sup.6                                                                             92.9                                          6.25                                                                             28     0     22       10.sup.5                                                                             78.6                                          3.67                                                                             7      1     1        10.sup.6 -10.sup.8                                                                   16.7                             __________________________________________________________________________     .sup.(a) Six daily intraperitoneal doses.                                

Table XXXII clearly shows the superiority of SPLV entrapped ticarcillinin clearing infection due to P. aeruginosa in a single daily doseregimen. The SPLV-entrapped ticarcillin was 30 times more effective thanfree drug alone.

                                      TABLE XXXIII                                __________________________________________________________________________    EFFECT OF SPLV-ENTRAPPED TOBRAMYCIN ADMINISTERED INTRA-                       PERITONEALLY ON P. AERUGINOSA PYELONEPHRITIS IN RATS                                                                MEAN                                    EXPERI-   I.P.               #        TITER OF                                                                             %                                MENTAL    PER DAY                                                                             # ANIMALS                                                                            #     CLEARED OF                                                                             INFECTED                                                                             CLEARED OF                       GROUP     (mg/kg)                                                                             IN GROUP                                                                             DEATHS                                                                              INFECTION                                                                              ANIMALS                                                                              INFECTION                        __________________________________________________________________________    CONTROLS  --    110    18    4        10.sup.5 -10.sup.6                                                                    4.3                             (INFECTED/                                                                    UNTREATED)                                                                    CONTROLS  --    7      0     N.A..sup.a                                                                              0     N.A..sup.a                       (UNINFECTED/                                                                  UNTREATED)                                                                    FREE      4     16     2     4        10.sup.4 -10.sup.5                                                                   28.6                             TOBRAMYCIN                                                                    IP (6 Dosages)                                                                SPLV-     4     40     3     37        0     100.00                           TOBRAMYCIN                                                                    (IP 6 Dosages)                                                                BUFFER    --    7      0     2        10.sup.5 -10.sup.6                                                                   28.5                             Filled SPLVs                                                                  IP (6 Dosages)                                                                BUFFER    4     7      0     1        10.sup.4                                                                             14.3                             Filled SPLVs and                                                              Free Tobramycin                                                               IP (6 Dosages)                                                                __________________________________________________________________________     .sup.(a) Not applicable.                                                 

Table XXXIII shows neither SPLVs alone nor SPLVs mixed with aqueoustobramycin were as effective in eliminating infection as tobramycinentrapped within SPLVs. The efficacy of SPLVs alone or SPLVs mixed withtobramycin were only as effective as aqueous tobramycin alone.

                                      TABLE XXXIV                                 __________________________________________________________________________    EFFECT OF SPLV-ENTRAPPED TOBRAMYCIN ADMINISTERED INTRA-                       VENOUSLY ON P. AERUGINOSA PYELONEPHRITIS IN RATS                                                                    MEAN                                    EXPERI-   I.V..sup.a         #        TITER OF                                                                             %                                MENTAL    PER DAY                                                                             # ANIMALS                                                                            #     CLEARED OF                                                                             INFECTED                                                                             CLEARED OF                       GROUP     (mg/kg)                                                                             IN GROUP                                                                             DEATHS                                                                              INFECTION                                                                              ANIMALS                                                                              INFECTION                        __________________________________________________________________________    CONTROLS  --    110    18    4        10.sup.5 -10.sup.6                                                                    4.3                             (INFECTED/                                                                    UNTREATED)                                                                    FREE      32    16     0     9        10.sup.5 -10.sup.6                                                                   56.2                             TOBRAMYCIN                                                                              16     7     1     1        10.sup.3 -10.sup.4                                                                   16.7                                        4    14     1     2        10.sup.5 -10.sup.6                                                                   15.4                             SPLV-     32    14     0     14        0     100.00                           TOBRAMYCIN                                                                              16    26     3     18        0     100.00                                      8    28     3     10       10.sup.4 -10.sup.5                                                                   40.0                                        4    35     7     6        10.sup.5 -10.sup.6                                                                   21.4                             __________________________________________________________________________     .sup.(a) One intravenous dose.                                           

Table XXXIV shows that SPLV entrapped tobramycin was three times moreeffective in eliminating infection for equivalent single doses whencompared with aqueous tobramycin administered intravenously.

                                      TABLE XXXV                                  __________________________________________________________________________    EFFECT OF SPLV-ENTRAPPED TOBRAMYCIN ADMINISTERED BY                           VARIOUS ROUTES ON P. AERUGINOSA PYELONEPHRITIS IN RATS                                                              MEAN                                    EXPERI-   DOSAGE.sup.a       #        TITER OF                                                                             %                                MENTAL    PER DAY                                                                             # ANIMALS                                                                            #     CLEARED OF                                                                             INFECTED                                                                             CLEARED OF                       GROUP     (mg/kg)                                                                             IN GROUP                                                                             DEATHS                                                                              INFECTION                                                                              ANIMALS                                                                              INFECTION                        __________________________________________________________________________    CONTROLS  --    110    18    2        10.sup.5 -10.sup.6                                                                    4.3                             (INFECTED/                                                                    UNTREATED)                                                                    FREE      16    7      1     1        10.sup.3 -10.sup.4                                                                   16.7                             TOBRAMYCIN                                                                    SPLV-                                                                         TOBRAMYCIN                                                                    Intravenous                                                                             16    28     4     22       10.sup.4                                                                             91.7                             Intraperi-                                                                              16    7      0     6        10.sup.2                                                                             85.7                             toneal                                                                        Subcutaneous                                                                            16    7      1     1        10.sup.4                                                                             16.7                             Oral      16    7      2     1        10.sup.5                                                                             20.0                             __________________________________________________________________________     .sup.(a) One dose.                                                       

Table XXXV demonstrates the efficacy of SPLV-entrapped tobramycinadministered by different routes. Intravenous and intraperitonealinjections of SPLV-entrapped tobramycin appear to be the mostefficacious.

15. EXAMPLE: ENHANCEMENT OF ANTIBACTERIAL ACTIVITY AGAINST CLOSTRIDIUMNOVYI USING SPLVS CONTAINING GENTAMICIN AND CLINDAMYCIN

In this example, the antibacterial activity and clinical effectivenessof various preparations of gentamicin (an aminoglycoside antibiotic) andclindamycin (a derivative of the amino acid trans-L-4-n prophylhygrinicacid attached to a sulfur-containing derivative of an octose) in thetreatment of anaerobic would infection of Clostridium novyi.

SPLVs containing gentamicin (SPLV/GENT) were prepared as described inSection 7.1 using 100 mg gentamicin. SPLVs containing clindamycin(SPLV/CLIN) were prepared the same way except that 100 mg clindamycinwas used in place of the gentamicin. SPLVs containing both gentamicinand clindamycin in one liposome preparation (SPLV/GENT-CLIN) wereprepared by the procedure described in Section 7.4 using 100 mg of eachantibiotic, gentamicin and clindamycin. All SPLV preparations werewashed three times in physiological saline.

15.1. Infection of Mice Using Clostridium Novyi

Twenty Swiss Webster adult female mice were injected in the right rearfootpad with 0.05 ml of a suspension of a Clostridium novyi prepared asfollows: C. novyi were grown for one day to stationary phase (10⁸ to 10⁹CFU/ml) in BHI media in an anaerobic blood bottle. The inoculum wasprepared by diluting the culture 1:100 using fresh degassed BHImedia;thus the inoculum contained approximately 10⁷ CFU/ml.

15.2. Treatment of Infected Mice

Twenty four hours after infection the mice were divided into 4 groups of5 mice each which were treated as follows: Group 1 (controls) receivedno treatment; Group 2 received SPLVs containing gentamicin (100 mggentamicin/kg body weight, I.P.); Group 3 received SPLVs containingclindamycin (100 mg clindamycin/kg body weight, I.P.); and Group 4received SPLVs containing both clindamycin and gentamicin in oneliposome preparation (100 mg of each antibiotic per kg body weight,I.P.). The diameters of the infected feet were measured using calipersand compared to control mice which were injected only with fresh media.Results are shown on Table XXXVI.

                                      TABLE XXXVI                                 __________________________________________________________________________    EFFECT OF SPLVS CONTAINING GENTAMICIN AND                                     CLINDAMYCIN ON CLOSTRIDIUM NOVYI INFECTION IN MICE                                    MEAN                                                                          FOOTPAD                                                                              SURVIVAL                                                               DIAMETER                                                                             DAYS POST INFECTION                                                                             %                                            GROUP   (INCHES)                                                                             1  2  3  4  5  6-19                                                                             SURVIVAL                                     __________________________________________________________________________    GROUP 1                                                                       Control 0.167  0/5                                                                              0/5                                                                              0/5                                                                              0/5                                                                              0/5                                                                              0/5                                                                              0%                                           Untreated                                                                     GROUP 2                                                                       SPLV/GENT                                                                             0.177  5/5                                                                              3/5                                                                              0/5                                                                              0/5                                                                              0/5                                                                              0/5                                                                              0%                                           GROUP 3                                                                       SPLV/CLIN                                                                             0.177  5/5                                                                              1/5                                                                              1/5                                                                              0/5                                                                              0/5                                                                              0/5                                                                              0%                                           GROUP 4                                                                       SPLV/   0.166  5/5                                                                              4/5                                                                              4/5                                                                              4/5                                                                              4/5                                                                              3/5                                                                              60%                                          GENT-CLIN                                                                     __________________________________________________________________________     .sup.1 The mean footpad diameter of uninfected mice inoculated with fresh     media is 0.119.                                                          

These results demonstrate that SPLVs containing both gentamicin andclindamycin in one liposome preparation were most effective in thetreatment of the anaerobic infection of the wounds.

It will be apparent to those skilled in the art that many modificationsandvariations may be made without departing from the spirit and scope ofthe invention. The specific embodiments described are given by way ofexample only and the invention is limited only by the appended claims.

What is claimed is:
 1. Stable plurilamellar vesicles comprising aplurality of lipid bilayers enclosing aqueous compartments containing atleast one entrapped solute, the concentration of such solute in eachcompartment being substantially equal to the concentration of soluteused to prepare the lipid vesicle, in which at least one antimicrobialagent is entrapped within the vesicles.
 2. The stable plurilamellarvesicles of claim 1 wherein said antimicrobial agent is selected fromthe group consisting of beta-lactam antibiotics, tetracyclineantibiotics, aminoglycoside antibiotics, macrolide antibiotics, andpolymyxin antibiotics.
 3. The stable plurilamellar vesicles of claim 2wherein said antimicrobial agent is a beta-lactam antibiotic selectedfrom the group consisting of penicillins and cephalosporins.
 4. Thestable plurilamellar vesicles of claim 2 wherein said antimicrobialagent is an aminoglycoside antibiotic selected from the group consistingof dihydrostreptomycin, streptomycin, gentamicin, kanamycin, andneomycin.
 5. The stable plurilamellar vesicles of claim 4 wherein saidaminoglycoside antibiotic is streptomycin.
 6. The stable plurilamellarvesicles of claim 4 wherein said aminoglycoside antibiotic isgentamicin.
 7. A method of treating infection in an animal, said methodcomprising administering a therapeutically effective amount of anantimicrobial agent contained in stable plurilamellar vesiclescomprising a plurality of lipid bilayers enclosing aqueous compartmentscontaining at least one entrapped solute, the concentration of suchsolute in each compartment being substantially equal to theconcentration of solute used to prepared the lipid vesicle.
 8. Themethod of claim 7 wherein the infection is intracellular.
 9. The methodof claim 7 wherein the infection is Brucella spp., Mycobacterium spp.,Salmonella spp., Listeria spp., Francisella spp., Histoplasma spp.,Corynebacterium spp., Coccidioides spp., Pseudomonas spp., orlymphocytic choriomeningitis virus.
 10. The method of claim 7 whereinsaid antimicrobial agent is selected from the group consisting ofbeta-lactam antibiotics, tetracycline antibiotics, aminoglycosideantibiotics, macrolide antibiotics, and polymyxin antibiotics.
 11. Themethod of claim 10 wherein said antimicrobial agent is an aminoglycosideantibiotic selected from the group consisting of dihydrostreptomycin,streptomycin, gentamicin, kanamycin, and neomycin.
 12. The method ofclaim 11 wherein the infection is Brucella spp., Mycobacterium spp.,Salmonella spp., Listeria spp., Francisella spp., Histoplasma spp.,Corynebacterium spp., Coccidioides spp., Pseudomonas spp., orlymphocytic choriomeningitis virus.
 13. The method of claim 12 whereinthe infection is intracellular.
 14. The method of claim 13 wherein theaminoglycoside antibiotic is gentamicin.
 15. The method of claim 14wherein the infection is Mycobacterium spp.
 16. The method of claim 13wherein the aminoglycoside antibiotic is streptomycin.
 17. The method ofclaim 16 wherein the infection is Brucella spp.